Recently, traces of double-positive FoxP3+RORγt+ T cells were identified and viewed as dual programming differentiation intermediates geared toward development into T regulatory or Th17 cells. In this study, we report that FoxP3+RORγt+ intermediates arise in the NOD mouse T cell repertoire prior to inflammation and can be expanded with tolerogen without further differentiation. Furthermore, FoxP3+RORγt+ cells express both CD62L and membrane-bound TGFβ and use the former to traffic to the pancreas and the latter to suppress effector T cells both in vitro and in vivo. The cells perform these functions as FoxP3+RORγt+ intermediates, despite being able to terminally differentiate into either FoxP3+RORγt− T regulatory or FoxP3−RORγt+ Th17 cells on polarization. These previously unrecognized observations extend plasticity to both differentiation and function and indicate that the intermediates are poised to traffic to sites of inflammation and target diverse pathogenic T cells, likely without prior conditioning by effector T cells, thus broadening efficacy against autoimmunity.
Naive CD4+ Th cells differentiate into one of several CD4+ T cell lineages including Th1, Th2, Th17, and T regulatory cells (Tregs) depending on environmental stimuli. This process offers flexibility that the type of Ag and the cytokine milieu exploit to coordinate measured immune responses (1). Lately, it has become clear that a delicate cellular and programming interplay between Tregs and Th17 cells dictates the balance between health and autoimmunity (2).
Naive Th cells stimulated with Ag in the presence of TGFβ upregulate expression of the transcription factor FoxP3 (3) and the phenotypic cell surface marker CD25 and develop into Tregs (4, 5). FoxP3 upregulation diverts the cell from activated to regulatory status by disrupting normal NFAT:AP-1 binding and instead forming an NFAT:Foxp3 complex (6). These cells display effective suppressive function against pathogenic self-reactive T cells (7–9). In contrast, when naive T cells are stimulated with Ag in the presence of both TGFβ and IL-6, they upregulate the transcription factor RORγt (10) and develop into Th17 cells (11, 12), a distinct lineage from Th1 and Th2 cells (13, 14) that produces the cytokine IL-17, hence the designation Th17 (15). Th17 cells proved pathogenic in several autoimmune disease models (16, 17), including type I (TID) or autoimmune diabetes (18).
Given that IL-6 diverts differentiation from Tregs to Th17 to give rise to cells with opposite function, emphasis was put on the plasticity of Th programming (19–22). Recently, it was reported that the differentiation of naive Th cells transits through a FoxP3/RORγt intermediate stage to provide further flexibility for the development of measured responses (2, 19). There is even evidence that fully differentiated Tregs can be reprogrammed to a Th17 phenotype (19), whereas fully differentiated Th17 cells can convert to Th1 lymphocytes (20–22). Because intermediates represent a transitional state, only traces of cells are available at any given time and isolating a sufficient number of cells to investigate molecular programs or in vivo function has not been feasible.
In this study, we were able to expand FoxP3+RORγt+ cells in vivo and demonstrate that these cells are fully functional. Indeed, Ig-GAD1, an immunoglobulin molecule genetically engineered to incorporate the glutamic acid decarboxylase (GAD) 524–543 diabetogenic aa sequence (designated GAD1) expands Tregs that protect against TID or autoimmune diabetes (23). Examination of these cells showed increased numbers of FoxP3+RORγt+ cells. Interestingly, these intermediates are present within the natural repertoire and express CD62L, which is required for trafficking as well as active membrane-bound TGFβ (mTGFβ), through which they suppress pathogenic T cells. Importantly, the FoxP3+RORγt+ Tregs did not secrete IL-17, in agreement with previously published results (24–26). However, under Th17-polarizing conditions, they were able to fully differentiate into RORγt+ cells capable of producing IL-17 cytokine. CD62L and TGFβ were expressed on the FoxP3+RORγt+ Tregs in the natural repertoire prior to disease onset, which likely guides the Tregs to the site of inflammation to target diverse effector T cells. These previously unrecognized findings indicate that FoxP3+RORγt+ intermediates are fully functional, broadening Th plasticity to both programming and function. Moreover, as the cells express CD62L and mTGFβ prior to inflammation, they do not require conditioning by effector cells but are able to traffic to sites of inflammation and target diverse T cell specificities.
Materials and Methods
NOD (H-2g7), NOD.scid, NOD.BDC2.5 (27), and NOD.FoxP3:GFP mice were used according to the guidelines of the University of Missouri of Columbia Animal Care and Use Committee. NOD.FoxP3:GFP reporter mice were generated by breeding C57BL/6.FoxP3:GFP knock-in animals (3) into NOD mice for 10 backcross generations.
Ig-GAD1 (23) is an Ig chimeras carrying GAD1 peptide corresponding to aa residues 524–543 (SRLSKVAPVIKARMMEYGTT) of GAD65 (28). This was done by inserting GAD1 nucleotide sequence into the H chain veriable region of 91A3 IgG2b molecule and transfecting the resulting 91A3H-GAD1 chimeric gene along with the parental 91A3 κ-chain gene into a non-Ig–secreting SP2/0 myleoma B cells (234)2SO4 as was previously described (29).
Expansion of FoxP3 expressing T cells by Ig-GAD1
NOD and NOD.FoxP3-GFP reporter mice are given i.p. 300 μg aggregated Ig-GAD1 in saline solution at week 4, 5, and 6. The mice are sacrificed at the end of week 6 that is 5 d after the last injection. For analysis of FoxP3 T cells at week 5, the mice receive two injections only, one at week 4 and one at week 5 and the animals are sacrificed 5 d later.
Assessment of diabetes
Assessment of blood glucose levels used Accu-Chek Advantage monitoring system. A mouse was considered diabetic when the blood glucose levels were >300 mg/dl for two consecutive measures.
Purification of pancreatic cells
Islets and infiltrating cells were purified according to a standard procedure (30
Neutralization of CD62L in vivo
Cells producing MEL-14 anti-CD62L Ab were grown large scale in DMEM media containing 10% iron-enriched calf serum (HyClone). Anti-CD62L Ab was then purified using columns containing CNBr-activated Sepharose linked to mouse anti-rat Ab. For neutralization of CD62L during expansion of FoxP3-expressing cells, 1 mg anti-CD62L Ab in PBS was administered i.p. along with each aggregated Ig-GAD1 injection. For neuralization of CD62L during transfer of FoxP3-expressing cells, 1 mg anti-CD62L Ab in PBS was administered i.p. along with the i.v. adoptive transfer of FoxP3int Tregs.
Flow cytometery analyses
Cell surface staining.
Splenocytes, pancreatic cells, pancreatic lymph node cells, or purified CD4+
FoxP3 and RORγt intracellular staining.
Cell analyses used a FACScan (Becton Dickson, San Jose, CA) or Cyan ADP (Dako, Denmark) flow cytometers and the FlowJo software (Tree Star, Ashland, OR). All staining data are representative of at least three replicates.
Suppression of passive diabetes by Tregs
The 10 million splenocytes from diabetic NOD mice were injected i.v. into NOD.scid recipient mice along with 5 × 105 of suppressor cells. FoxP3int, FoxP3hi, and FoxP3− T cells were sorted to 95% purity based on GFP expression from NOD FoxP3:GFP reporter mice on a MoFlo cell sorter (Dako).
This was performed as previously described (23). Briefly, allogenic effector NOD T cells were incubated in a 96-well plate (200,000/well) with CD11c+ C57BL/6 APC (100,000/well) that were purified from bulk splenocytes on anti–CD11c-coupled Miltenyi microbeads. Sorted (95% purity) FoxP3int or FoxP3hi Tregs were added at the indicated ratios and the culture was incubated for 3 d with the addition of 1 μCi [3H]thymidine during the last 8 h of culture. The cells were then harvested on a Trilux 1450 Microbeta Wallac Harvester, and incorporated [3H]thymidine was counted using the Microbeta 270.004 software (Wallac, Waltham, MA).
T cell activation and polarization
Purified T cells were incubated in a 96-well round-bottom plate for 72 h along with plate-bound anti-CD3 (2C11) (10 μg/ml), soluble anti-CD28 (PV1) (1 μg/ml) and 100 U rIL-2 (212–12; PeproTech, Rocky Hills, NJ).
Purified T cells were incubated under activating conditions and polarized with 10 ng/ml rIL-12 (210–12; PeproTech) and 10 μg/ml anti–IL-4 (11B11).
Purified T cells were incubated under activating conditions and polarized with 3 ng/ml rhTGFβ (100–21C; PeproTech), 10 ng/ml rIL-6 (216–16; PeproTech), 10 μg/ml anti-IFNγ (R4-6A4), and 10 μg/ml anti–IL-4 (11B11).
Purified T cells were incubated under activating conditions and polarized with 3 ng/ml rhTGFβ (100–21C; PeproTech), 10 μg/ml anti-IFNγ (R4–6A4), and 10 μg/ml anti–IL-4 (11B11).
After 5 d polarization, the T cells were stimulated for 6 h with 50 ng/ml PMA and 500 ng/ml ionomycin in the presence of brefeldin A. The cells were then permeabilized with 0.2% saponin and intracellular cytokines were detected by staining with anticytokine Abs.
Detection of cytokine in culture supernatant used ELISA as described (18, 23, 29). Also, cytokine secretion was detected on the cells surface to identify IL-10–producing cells using Miltenyi bioconjugate mouse IL-10 secretion assay according to the manufacturer’s instruction (Miltenyi Biotec, Bergisch Gladbach, Germany).
FoxP3hi or FoxP3int
The p values were calculated using the two-tailed Student t test.
Protective Ag therapy against TID expands discrete FoxP3 Tregs
We have previously described a protective treatment regimen with Ig-GAD1, which expands splenic Tregs when administered into NOD mice at 4–6 wk of age (23). The same regimen, however, was unable to protect against TID when administered at 8 wk of age, despite effective expansion of splenic Tregs (23) (Supplemental Fig. 1A). The 8-wk-old splenic Tregs, however, had diminished levels of mTGFβ (Supplemental Fig. 1B), which is indispensable for suppression of the disease (23).
To gain further insight on the specific loss of mTGFβ+ Tregs during the transition from 6–8 wk, which coincides with ongoing progressive insulitis in NOD mice, we opted to compare the phenotype of the two populations. The initial experiments were focused on FoxP3 protein expression in 5-, 6-, 7-, and 8-wk-old splenic Tregs from Ig-GAD1–treated mice. Much to our surprise, these splenic Tregs comprised two distinct populations with one displaying FoxP3int expression and the second with FoxP3hi expression (Fig. 1A). Intriguingly, there was a gradual decrease of the FoxP3int population and, by week 8, most of the Tregs had a FoxP3hi phenotype (Fig. 1A). Indeed, 5-wk-old Tregs comprised 36% FoxP3int and 43% FoxP3hi cells. However, by 8 wk of age, 64% of Tregs were FoxP3hi and the FoxP3int population was significantly reduced to 6.3%. Interestingly, the decrease in FoxP3int Tregs coincides with the loss of mTGFβ in Ig-GAD1–expanded 8-wk-old splenic Tregs (23), which prompted us to determine the dynamics of mTGFβ expression by the two populations. The results illustrated in Fig. 1B clearly show that mTGFβ was predominantly expressed on the FoxP3int population but was significantly reduced when the FoxP3int cells were no longer detectable by week 8. Indeed, the FoxP3hi population, which represented 39% and 52% of total Tregs on week 5 and 8, respectively, had no significant mTGFβ expression at any time point. However, the FoxP3int mTGFβ+ population went down from 15% of total Tregs at week 5 to 4% by week 8 (Fig. 1B). Similar patterns of FoxP3 and TGFβ expression was observed in untreated mice (Supplemental Fig. 2). In addition, a control Ig-hen egg lysozyme molecule, incorporating the I-Ag7–restricted nondiabetogenic hen egg lysozymepeptide instead of GAD1, was unable to expand any Treg cells in this model (23).
To determine which of the two populations (FoxP3int and FoxP3hi) might be involved in protection against diabetes during treatment with Ig-GAD1, we searched for Tregs in both the spleen and pancreas at week 6 and 8, time points where insulitis is progressive. As illustrated in Fig. 1C, FoxP3int Tregs dropped from 22 × 104 (per 106 counted CD25+ cells) at week 6 to 4 × 104 at week 8 in the spleen but were maintained at high numbers in the pancreas (27 and 22 × 104 per 106 counted CD25+ cells at weeks 6 and 8, respectively). Interestingly, FoxP3hi Tregs were present at high numbers in the spleen at both week 6 and 8 (39 and 50 × 104 per 106 CD25+ counted cells, respectively) but were minimal in the pancreas at both time points (Fig. 1C). These results indicate that Ig-GAD1 expands phenotypically distinct Tregs that reside in different organs during the onset of disease. In untreated mice FoxP3int and FoxP3hi T cells were present, although at smaller numbers, than Ig-GAD1–treated mice (Supplemental Fig. 2B, 2C). Furthermore, their residential patterns at weeks 6 and 8 of age were similar to Ig-GAD1–expaneded T cells, possibly indicating that both FoxP3int and FoxP3hi T cells arise in the normal repertoire and can be expanded by Ig-GAD1.
FoxP3int T cells express the Th17 signature RORγt transcription factor
Given their differential expression of mTGFβ and location during insulitis, we sought to further characterize the two populations of Tregs for expression of trafficking molecules, transcription factors, and effector cytokines. We performed these analyses at 6 wk of age in the spleen, before the cells begin trafficking and become subject to local environmental changes. Fig. 2 shows that 73.2% of CD4+CD25+ FoxP3int cells express mTGFβ, whereas only 12.4% of the CD4+CD25+FoxP3hi Tregs had mTGFβ (Fig. 2A). Given that the two populations had different localization patterns during progressive insulitis and that CD62L is known to regulate Treg trafficking and function in NOD mice (31–33), the cells were tested for CD62L expression. Not surprisingly, 87.3% of splenic FoxP3int Tregs expressed CD62L, whereas only 3.2% of FoxP3hi Tregs had CD62L. These findings may explain the presence of FoxP3int but not FoxP3hi Tregs in the pancreas during insulitis (32). GITR protein was, however, expressed to a similar extent on both FoxP3int and FoxP3hi Tregs (92.5% and 93.8%, respectively) (Fig. 2A).
The most surprising finding in these analyses is that FoxP3int mTGFβ+CD62L+ but not FoxP3hi mTGFβ−CD62L− cells had an impressive level of the Th17 signature transcription factor RORγt (Fig. 2A). Indeed, 77.6% of FoxP3int cells expressed RORγt protein, whereas FoxP3hi cells had no detectable RORγt. FoxP3+RORγt+ double-positive T cells have been described recently and were thought to represent transitional differentiation intermediates geared toward single-positive RORγt+ Th17 or FoxP3+ Tregs (2, 19). These intermediates did not produce IL-17 under nonpolarizing conditions in vitro (Supplemental Fig. 3), which is in good agreement with reports showing that FoxP3+RORγt+ intermediates are unable to secrete IL-17 because of inhibition by FoxP3 (24, 25).
The two other molecules that were analyzed were ICOS and IL-10. Similar to what was observed in humans (34), FoxP3int mTGFβ+ Tregs express lower levels of ICOS (33.2%) than FoxP3hi TGFβ− cells (63.8%) (Fig. 2A). Also, in agreement with what has been demonstrated in human Tregs (34), FoxP3hi cells, which are highly ICOS+ (Fig. 2A), secrete greater amounts of IL-10 (75.9%) than their FoxP3int counterparts (29.9%) (Fig. 2A). Overall, the FoxP3int RORγt+ intermediates express CD62L and mTGFβ, whereas the FoxP3hi Tregs express ICOS and produce IL-10 and both populations have GITR (Fig. 2A).
Given that both the FoxP3int RORγt+ intermediates and FoxP3hi Tregs express suppressive molecules, it is possible that both could display suppressive function in vitro. This premise was tested in an allogenic system where effector NOD cells were incubated with C57BL/6 dendritic cells in the presence or absence of highly purified FoxP3int RORγt+ intermediates or FoxP3hi Tregs and proliferation of effector T cells was measured. The results indicated that both populations displayed significant suppression of NOD effector T cell responses in vitro (Supplemental Fig. 4).
Splenic FoxP3int RORγt+ T cells protect against passive TID
The mTGFβ serves as a suppressive molecule on Tregs (23, 35–37). In fact, disease suppression by Ig-GAD1–expanded Tregs was dependent on mTGFβ as a blocking anti-TGFβ Ab abrogated protection (23). Before testing the in vivo function of highly purified FoxP3int cells, we used the NOD.FoxP3-GFP reporter mouse and ensured that Ig-GAD1 treatment expands these intermediates and yields sufficient cells for testing against diabetes. Subsequently, the mice were treated with Ig-GAD1 and FoxP3−, FoxP3int (which highly coexpress RORγt), or FoxP3hi (RORγt−) T cells were sorted on the basis of FoxP3 expression. To test for protection against diabetes, the sorted cells were adoptively transferred into NOD.scid recipient mice along with splenocytes from recently diabetic mice. Blood glucose levels were then monitored for 6 wk posttransfer. The results indicate that by week 6 after transfer, 100% of the mice that received the control FoxP3− T cells were diabetic (Fig. 2B). Mice recipient of both FoxP3int and FoxP3hi Tregs showed protection from diabetes, as 70% and 30% were protected from disease by week 6, respectively (Fig. 2B). Statistical analyses indicated that FoxP3int display significant protection relative to FoxP3− T cells (p = 0.0016), whereas FoxPhi were less protective at this time point after transfer (p = 0.0778). Overall, this data indicates that FoxP3int RORγt+ intermediates, which are found in the pancreas during the onset of insulitis, exercise suppressive function in vivo, to a better extent than FoxP3hi counterparts.
Ig-GAD1 treatment sustains migration of FoxP3int RORγt+ T cells to the pancreas during insulitis
To protect against passive or spontaneous diabetes, splenic FoxP3int Tregs likely migrate to the pancreatic lymph node and the pancreas, where they can suppress effector cells (Fig. 1C). Because these cells express RORγt (Fig. 2A) there is potential to exercise their function as FoxP3int RORγt+ intermediate cells. Alternatively, the cells could complete their differentiation on the way to the inflammatory site and perform suppression as fully differentiated FoxP3int RORγt– Tregs. To test this premise, we began by analyzing the dynamics of the FoxP3 cells during the onset of insulitis. The results indicate that in the Ig-GAD1–treated mice the FoxP3int cells are located in both the pancreatic lymph node (Fig. 3A) and pancreas (Fig. 3C) at week 6 as well as week 8 of age. However, in untreated mice, the cells are found in the pancreatic lymph node and pancreas at week 6 but by week 8 only a very small number reside in the pancreatic lymph node and the majority of the cells are located in the pancreas (Fig. 3B, 3D). Quantification of FoxP3+mTGFβ+ T cell numbers indicated that Ig-GAD1 therapy increases the frequency of these cells both in the pancreatic lymph node and pancreas during progressive insulitis relative to untreated mice (Fig. 3E, 3F). Indeed, the frequency of FoxP3+ mTGFβ+ Tregs at week 8 of age increased from 0.5 × 104 per 106 CD4+ T cells in the untreated mice to 2.4 × 104 per 106 CD4+ T cells in Ig-GAD1–treated animals. The accumulation of these suppressive cells at the site of inflammation likely suggests a protective role against diabetes and offers a mechanism by which Ig-GAD1 therapy delays the disease (23).
Pancreatic FoxP3int RORγt+ T cells protect against disease without further differentiation
To determine whether these regulatory cells locate to the site of inflammation and perform suppressive function as FoxP3int intermediates bearing RORγt or rather as fully differentiated FoxP3int RORγt− Tregs, we tested the pancreatic lymph node and pancreas resident FoxP3int TGFβ+ cells for expression of RORγt at week 6 and 8 of age in both un-treated as well as Ig-GAD1–treated NOD mice. The findings indicate that, in the Ig-GAD1–treated mice, the majority of FoxP3int mTGFβ+ Tregs strongly maintain their RORγt+ phenotype both in the pancreatic lymph node (97.5% and 95.3% at weeks 6 and 8, respectively) (Fig. 4A) and the pancreas (97.7% and 98.1% at weeks 6 and 8, respectively) (Fig. 4B). No detectable single-positive FoxP3int RORγt− cells were found in either organ, indicating that conversion among the TGFβ+ Treg population was minimal. Similarly, in the untreated mice, 97.7% of the pancreatic lymph node (Fig. 4A) and 91.9% of the pancreas (Fig. 4B) FoxP3int mTGFβ+ Tregs express RORγt at week 6 with no detectable single-positive FoxP3int RORγt− cells. Also, at week 8 when FoxP3int mTGFβ+ Tregs were minimally present in the pancreatic lymph node (Fig. 3B) the majority (95.9%) of the cells available in the pancreas express RORγt, indicating that conversion was not operative. These results indicate that FoxP3int TGFβ+ T cells express RORγt and maintain their intermediate (FoxP3int TGFβ+ RORγt+) phenotype upon migration to the inflammatory sites.
The accumulation of FoxP3int TGFβ+RORγt+ intermediates in the pancreas of mice treated with Ig-GAD1, which are protected from diabetes, suggest that these cells are potentially functional and contribute to resistance against the disease. To test this premise, FoxP3int Tregs were purified from the pancreas of 6- and 8 wk-old mice on the basis of GFP expression and transferred to NOD.scid recipients, along with diabetogenic splenocytes, and the recipients were monitored for blood glucose levels. The results indicated that 6- as well as 8-wk-old pancreatic FoxP3int TGFβ+RORγt+ Tregs were strongly protective as 70% (Fig. 4C) and 80% (Fig. 4D) of the mice were protected from diabetes, respectively, whereas all (100%) of the control mice without Treg transfer developed diabetes (Fig. 4E). To determine whether the cells were further differentiated in the recipient mice, we harvested the spleens, pancreatic lymph nodes, and pancreas from the hosts and analyzed the cells for FoxP3 and RORγt expression. As indicated in Fig. 4E, 93.7% of FoxP3int cells in the spleen express RORγt. Interestingly, the 99.3% of the FoxP3int cells that migrated to the pancreatic lymph node also maintain expression of RORγt (Fig. 4E, median panel). Equally important, 89.4% of the FoxP3int cells that migrated to the pancreas still coexpress RORγt (Fig. 4E, right panel). Overall, these findings indicate that FoxP3int TGFβ+RORγt+ cells migrate to the site of inflammation and protect against diabetes without loss of RORγt or full differentiation to FoxP3+RORγt− Tregs.
CD62L is required by FoxP3int RORγt+ Treg for migration to the pancreas and protection against diabetes
Both FoxP3hi and FoxP3int Tregs are present in the spleen, but only FoxP3int express CD62L (Fig. 2A) and migrate to the pancreas (Fig. 1C). Given that CD62L was previously shown to facilitate trafficking of Tregs to the pancreatic lymph node (32), it is possible that FoxP3int cells preserve CD62L expression to migrate to the site of inflammation and confer resistance against diabetes. To address this point, we began by determining whether Ig-GAD1–expanded FoxP3int RORγt+ cells that migrate to the pancreas maintain CD62L expression. The results presented in Fig. 5A indicate that pancreatic FoxP3int RORγt+ Tregs maintain high levels of CD62L expression at both week 6 and week 8 of age in untreated as well as Ig-GAD1–treated mice. Indeed, at 6 wk of age, 89% of FoxP3int Tregs from both Ig-GAD1–treated and -untreated mice express CD62L (Fig. 5A). Similarly, at 8 wk, most (86%) of the cells preserved CD62L expression in Ig-GAD1–treated mice, whereas 78% had CD62L in the untreated mice (Fig. 5A). This data suggests that FoxP3int TGFβ+RORγt+ Tregs might rely on CD62L to traffic to the pancreas, the site of inflammation in the NOD mouse model. To determine whether CD62L is required for migration of FoxP3int TGFβ+RORγt+ T cell to the pancreas, mice were given Ig-GAD1 therapy (to expand Tregs) accompanied with a neutralizing anti-CD62L Ab and migration of the cells to the pancreas was evaluated. The results in Fig. 5B indicate that in vivo neutralization of CD62L retains FoxP3int Tregs in the SP that can no longer migrate to the pancreas (Fig. 5B). This indicates that CD62L is required for trafficking of FoxP3int RORγt+ Tregs to the site of inflammation.
To determine whether CD62L-mediated trafficking of FoxP3int RORγt+ Tregs to the pancreas is required for protection against diabetes, NOD.scid mice were adoptively transferred with pancreatic FoxP3int RORγt+ Tregs along with diabetogenic splenocytes and the hosts were given anti-CD62L Ab or an isotype control. The mice were then monitored for blood glucose levels. The results show that neutralization of CD62L nullifies the suppressive function of FoxP3int RORγt+ Tregs as the mice were no longer protected against diabetes (Fig. 5C). Indeed, 80% of the mice given anti-CD62L were diabetic by week 6 postcell transfer (Fig. 5C, top panel). However, all mice (100%) recipient of rat IgG instead of anti-CD62L remained free from diabetes (Fig. 5C, median panel). Neutralization of CD62L protein had no effect on the ability of effector diabetogenic splenocytes to induce TID in this adoptive transfer model. This conclusion is drawn from the observation that transfer of diabetic splenocytes without FoxP3int RORγt+ Tregs, leads to diabetes when the mice are given anti-CD62L Ab. In fact, 100% of the mice in this control group became diabetic by week 6 postcell transfer (Fig. 5C, bottom panel), which parallels observations reporting that diabetogenic T cells enter the pancreas through an l-selectin independent pathway (38). Although, other studies have shown that anti-CD62L Ab can interfere with migration of diabeteogenic T cells and protect against diabetes (39). This, however, required frequent injection of a higher dose of the Ab and used wild-type NOD rather than NOD.scid mice. Overall, the results demonstrate that FoxP3int RORγt+ Tregs require CD62L to traffic to the pancreas and perform suppressive function, which bodes well with reports indicating that NOD.CD62L−/− animals develop robust TID (40).
FoxP3int RORγt+ T cells retain potential for differentiation into either Th17 or FoxP3hi Tregs
FoxP3int T cells, despite their high coexpression of RORγt, function as Tregs because they suppress effector T cells in vitro, migrate to the pancreas in vivo and confer resistance against passive diabetes. The question then is whether these cells represent a new subset of Tregs with a fixed phenotype or retain RORγt to preserve program plasticity with potential for differentiation into Th17 cells as was previously suggested (19). To test this premise, we sought to determine whether FoxP3int Tregs could be polarized toward a Th1, Th17, or FoxP3hi Treg phenotype. Accordingly, FoxP3int and FoxP3hi Tregs were sorted from NOD.FoxP3-GFP reporter mice and expression of FoxP3 was analyzed prior to investigation of polarization. Fig. 6A shows that the two populations had the expected FoxP3 level and were highly pure. Subsequently, the cells were polarized under Th1 (Fig. 6B), Th17 (Fig. 6C), and Treg (Fig. 6D) conditions and analyzed for IL-17 and IFNγ cytokine production. The results show that the FoxP3int population, which strongly expresses the transcription factor RORγt, could easily be polarized toward a Th17 phenotype as 37% of the cells produced high levels of IL-17 cytokine (Fig. 6C). Interestingly, this population was unable to differentiate toward a Th1 phenotype as measured by IFNγ production and no detectable cells were positive for this cytokine (Fig. 6B). Under Treg polarizing conditions, FoxP3int Tregs did not secrete significant levels of IL-17 or IFNγ (0.91%) (Fig. 6D). FoxP3hi Tregs appear to be terminally differentiated and were unable to produce IL-17 or IFNγ under Th1, Th17 or Treg-polarizing conditions (Fig. 6B–D). It seems, then, that Tregs that highly express FoxP3 are unable to polarize toward Th17 and the earlier reports of Treg to Th17 polarization may have included cells expressing low level of FoxP3 Tregs than can convert to T17 cells (19).
The FoxP3int cells were further analyzed for FoxP3 and RORγt mRNA on polarization to Tregs and Th17 cells. On Treg polarization, FoxP3 mRNA levels rose from 1 relative quantity (RQ) prepolarization to 5 RQ after polarization, whereas RORγt remained the same (Fig. 6E). However, under Th17 polarizing conditions, whereas FoxP3 was similar pre- and postpolarization, RORγt rose from 1 to 4.1 RQ pre- to postpolarization (Fig. 6E). Similar results were obtained at the protein level (not shown). Overall, these findings indicate that the FoxP3int RORγt+ population represents a differentiation intermediate stage that can further develop into either FoxP3hi Tregs or Th17 cells.
FoxP3int RORγt intermediate Tregs arise in the periphery
To better understand the origin of these suppressive FoxP3int RORγt+ Tregs, we sought to determine whether this population originates from the thymus in its FoxP3int form or arises in the periphery. To address this question, thymi from 6-wk-old NOD mice were harvested and FoxP3 protein level was measured on the CD4+CD25+ population, which represents 3.3% of all CD4+ single-positive T cells (Fig. 7A). The majority (76.5%) of these cells were FoxP3hi cells with the rest not expressing FoxP3 and FoxP3int could be observed. Further phenotypic analyses indicated that these thymic FoxP3 cells did not express RORγt, mTGFβ, or CD62L (Fig. 7B). These results indicate that the FoxP3int RORγt+ cells, which are observed in the spleen, pancreatic lymph node, and pancreas do not develop their FoxP3int RORγt+ phenotype while in the thymus but rather in the periphery, which is in agreement with the idea that this population is a differentiation intermediate of peripheral CD4+ T cells (2, 19).
The notion of Th cell plasticity (2, 19–22) suggests that the immune response is far more adaptable than was previously thought and is therefore able to respond more appropriately to environmental stimuli. Initially, the naive Th cell was viewed as a pluripotent precursor that could engage in one of multiple pathways and differentiate into a terminal Th1, Th2, Th17, or Treg cell (1). More recently, however, observations were made indicating that fully differentiated Tregs can reverse into Th17 cells (19) and a Th17 cell can switch to a Th1 phenotype (20–22). Evidence is accumulating indicating that Th plasticity is even broader and Th precursors engage in more than one pathway during differentiation, generating intermediate phenotypes susceptible for further programming (19, 41, 42). However, because intermediate cells are thought of as having a transient phenotype available only at low frequency, analysis of the mechanism underlying the mixed phenotypes or their function was limited. In this study, we identified a FoxP3int RORγt+ intermediate population that arises in the natural T cell repertoire alongside FoxP3hi (RORγt−) Tregs that could be expanded without further differentiation. These cells do not represent artifacts of the NOD.FoxP3-GPF reporter mice because they are expandable with Ig-GAD1 in normal NOD mice and display similar residential pattern in both strains (Supplemental Fig. 5). The cells arise in vivo before and after expansion with Ig-GAD1 and evidence is provided indicating that they express mTGFβ that serves for suppression of effector T cells (Fig. 1B, Supplemental Fig. 2) and CD62L that facilitates trafficking to sites of inflammation (Fig. 5A–C). In the pancreas, which is the site of inflammation in NOD mice, the cells retained the FoxP3int RORγt+ intermediate phenotype (Fig. 4A, 4B), as well as CD62L (Fig. 5A) and TGFβ (Fig. 4A, 4B), likely suggesting that they contribute to protection against the disease as intermediate cells. In fact, when the expanded cells were isolated from both the spleen and the pancreas and transferred to NOD.scid mice along with diabetogenic splenocytes, they were able to traffic again to the pancreatic lymph node and pancreas in the host animals (Fig. 4E) and confer resistance against diabetes (Fig. 4C, 4D). Surprisingly, on migration to the pancreas the cells did not downregulate RORγt and protected against disease as FoxP3int RORγt+ bearing both CD62L and TGFβ. However, if the cells are subject to polarization, they retain the ability to fully differentiate to Th17 cells and produce IL-17 cytokine (Fig. 6C), or Tregs with higher FoxP3 but significantly reduced RORγt (Fig. 6). It was previously shown that CD4+ T cells expressing CD62L protect against diabetes (43, 44). These suppressor cells may represent classical Tregs that use CD62L to traffic to the site of inflammation. However, it is not clear whether they display the differentiation intermediate phenotype.
Because CD62L and TGFβ were expressed on the intermediate Tregs in the natural repertoire prior to inflammation, it is unlikely that they were induced by factors related to effector T cells. Rather, it would be more logical to envision that they arise prior to the onset of disease to guide the Tregs to the site of inflammation and target the local effector T cells regardless of their phenotype. This indeed provides a functional plasticity that would add flexibility relative to effector dictated specific trafficking and suppression reported recently (41, 42).
Overall, the findings reported in this study indicated that Th intermediates display both differentiation (1, 19–22, 45) and functional plasticity that could sustain measured but broad effectiveness against autoimmunity.
Disclosures The authors have no financial conflicts of interest.
This work was supported by National Institutes of Health Grants RO1DK65748 and R21AI68746 (to H.Z.). D.M.T. and J.A.C. were supported by Life Sciences Fellowships from the University of Missouri, Columbia. C.M.H. was supported by National Institute of General Medical Sciences training Grant GM008396.
The online version of this article contains supplemental material.
Abbreviations used in this paper:
- glutamic acid decarboxylase
- membrane-bound active TGFβ
- relative quantity
- type I diabetes
- T regulatory cells.
- Received October 13, 2009.
- Accepted January 21, 2010.
- Copyright © 2010 by The American Association of Immunologists, Inc.