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. Author manuscript; available in PMC: 2012 Mar 25.
Published in final edited form as: Immunity. 2011 Mar 25;34(3):422–434. doi: 10.1016/j.immuni.2011.03.002

CD4+ CD25+ Foxp3+ regulatory T cells promote Th17 cells in vitro and enhance host resistance in mouse Candida albicans Th17 cell infection model

Pushpa Pandiyan 1, Heather R Conti 2, Lixin Zheng 1, Alanna C Peterson 3, Douglas R Mathern 1, Nydiaris Hernández-Santos 3, Mira Edgerton 2, Sarah L Gaffen 2,3, Michael J Lenardo 1,*
PMCID: PMC3258585  NIHMSID: NIHMS348343  PMID: 21435589

SUMMARY

Th17 cells and CD4+CD25+Foxp3+ regulatory T (Treg) cells are thought to promote and suppress inflammatory responses, respectively. Here we explore why under Th17 cell conditions, Treg cells did not suppress, but rather up-regulated the expression of interleukin-17A (IL-17A), IL-17F and IL-22 from responding CD4+ cells (Tresp). Up-regulation of IL-17 cytokines in Tresp cells was dependent on consumption of IL-2 by Treg cells especially at early time points both in vitro and in vivo. During an oral Candida albicans infection in mice, Treg cells induced IL-17 cytokines in Tresp cells, which markedly enhanced fungal clearance and recovery from infection. These findings show how Treg cells can promote acute Th17 cell responses to suppress mucosal fungus infections and reveal that Treg cells have a powerful capability to fight infections besides their role in maintaining tolerance or immune homeostasis.

INTRODUCTION

CD4+CD25+Foxp3+ T cells, termed T regulatory (Treg), cells are thought to be a stable lineage of cells that plays an active suppressive role in the maintenance of immunological self-tolerance and immune homeostasis, but whose role in protective immunity is not fully understood (Sakaguchi et al., 2009). The suppressive functions are exhibited in Foxp3 deficient mice and the human "immune dysregulation enteropathy polyendocrinopathy X-linked” (IPEX) syndrome patients that succumb to fatal inflammatory disorders associated with fewer numbers of Treg cells (Ochs et al., 2007). Interestingly, although IPEX patients manifest apparent autoimmune diseases, they also have a susceptibility to specific infectious diseases, notably Candida (C.) albicans infections, suggesting selective immunodeficiency (Ochs et al., 2007). The transcriptional repressive effects of the forkhead box P3 (Foxp3) protein render Treg cells incapable of producing certain key cytokines such as interleukin-2 (IL-2) and so they require an exogenous supply of these cytokines for their peripheral maintenance (Pandiyan and Lenardo, 2008). Indeed, Treg cells compete for IL-2 and other survival cytokines leading to cytokine deprivation apoptosis of effector T cells (Pandiyan et al., 2007). Careful experimental modeling of the cytokine competition mechanism of suppression by Treg cells reveals that suppression depends strongly on the local cytokine milieu and the proximity of Treg cells to effector cells during an immune response (Busse et al., 2010; Tang and Bluestone, 2008). Treg cells may not effectively suppress by cytokine competition when cytokines are abundant such as during an infection. Some studies have predicted that Treg cells could lose their suppressive functions during acute inflammation in microbial infection models (Oldenhove et al., 2009; Tsuji et al., 2009). Plasticity of Treg cells and their potential non-suppressive immune functions have been the recent focus of speculation (Zhou et al., 2009). Importantly, certain investigations have demonstrated protective functions for Treg cells during viral infections (Lanteri et al., 2009; Lund et al., 2008). Thus, whether Treg cells may have broader roles in immunity than just the previously recognized suppressor functions, is a key area for further discovery.

T helper-17 (Th17) cells produce abundant inflammatory cytokines and are key mediators in host defense, inflammatory disorders and autoimmune conditions (Korn et al., 2009). Mechanisms of interactions between Treg cells and Th17 cells, and the paradoxical ability of Treg cells to augment IL-17A induction are not well understood in vivo (Veldhoen et al., 2006; Xu et al., 2007). Therefore, we chose to study Treg cell function in the context of differentiating Th17 cells. One of the most important functions of Th17 cells in host immunity is to protect against fungal infections. Oropharyngeal candidiasis or “thrush”, an Acquired immune deficiency syndrome-defining illness is an infection by the commensal fungus C. albicans (Conti et al., 2009). It has been well documented in mice and humans, that Th17 cells and IL-17 production are critical for oral fungicidal immune responses by recruiting neutrophils to the oral mucosa and inducing salivary antimicrobial factors (Conti et al., 2009; Curtis and Way, 2009; Eyerich et al., 2008). Patients with hyper IgE syndrome with fingernail candidiasis or chronic mucocutaneous candidiasis have impaired Th17 cell responses (Milner et al., 2008). Interestingly, patients lacking Treg cells, including IPEX patients, those with IPEX-like syndrome (CD25 deficient patients) or Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy (APECED) patients deficient in the Autoimmune regulator (AIRE) protein also are highly susceptible to C. albicans infections (Kekalainen et al., 2007; Roifman, 2000). The underlying mechanism and possible roles of Treg cell deficiency in this susceptibility are unclear (Coutinho and Carneiro-Sampaio, 2008; Kekalainen et al., 2007; Ochs et al., 2009). Therefore, we chose to investigate the function of Treg cells in modulating Th17 cell responses in an oral C. albicans infection model.

Here we showed that Treg cells can powerfully promote the transition of naïve CD4 cells to Th17 cells producing the full suite of characteristic cytokines independently of the effect mediated by transforming growth factor (TGF)-β (Veldhoen et al., 2006; Xu et al., 2007). Treg cells achieved this by consuming IL-2, and thereby preventing it from inhibiting Th17 cell differentiation both in vitro and in vivo. Treg cells did not suppress, but actually promoted IL-17A-dependent clearance of fungi during acute C. albicans infection. However, despite contributing potently to this acute immunoprotective effect, Treg cells exhibited suppressive properties at late times and inhibited chronic Th17 cell mediated inflammatory bowel disease (IBD). Thus, we provide new insights into a new facet of Treg cell biology and that, in addition to immune suppression, they promote Th17 cell differentiation and participate in host protective immunity against fungal infections such as by C. albicans.

RESULTS

Treg cells upregulate Th17 cell cytokines from responder T (Tresp) cells in vitro

Previous studies have shown that TGF-β provided by Treg cells is essential for IL-17A induction in naïve CD4 cells stimulated with dendritic cells and IL-6 (Veldhoen et al., 2006; Xu et al., 2007). In order to study how Treg cells contributed to the induction of IL-17A in CD4 cells using Th17 cell polarizing conditions (Korn et al., 2009), we stimulated CD4+ CD44low CD62Lhigh CD25− naïve cells (Tresp), in the presence of control green fluorescent protein (GFP)− CD4+CD44lowCD25− (>99% purity) (Tcon) or GFP+ CD4+CD25+ Treg cells (>99% purity) flow cytometrically sorted from Foxp3gfp reporter mice. Tcon or Treg cells were derived from CD45.2 B6 mice and Tresp (equivalent to Tcon) cells were derived from CD45.1 congenic mice so that Tresp cells could be selectively identified using CD45.1 staining (Fig. S1). Under optimal Th17 cell polarizing conditions, we were surprised to observe that the fraction of IL-17A producers, as assessed by intracellular staining and the mean fluorescence intensity (MFI) of IL-17A in Tresp cells was still significantly boosted by co-cultivation with Treg cells compared to those cultured alone or with Tcon cells on day 3 (d3) (Fig. 1A). We confirmed these findings using flow cytometery sorted CD4+CD25+Treg cells (>99% purity) and CD4+CD44lowCD25−Tcon cell preparations in five independent experiments (Fig.1B, 1D (top panel)). Tresp cells co-cultured with Treg cells increased the frequency of IL-17A producers also at time points as late as day 7 (Fig. 1D (bottom panel), Fig. S2A). CD3–CD28 restimulated Tresp cells also showed increased IL-17A in Treg cell co-cultures compared to controls (S2B). Induction of IL-17A was a specific property of CD25+ Foxp3+ Treg cells because activated CD25+ effector (CD4+CD25+T-eff) cells did not up-regulate IL-17A in naïve Tresp cells (S2C). We also found that cytokines such as IL-17F and IL-22 were boosted by the presence of Treg cells indicating that the full differentiation program of Th17 cells was being promoted both on d4 and d6 (Fig.1C, 1E, Fig. S2D). Among Tresp cells, there were very few Foxp3+ and IFN-γ+ cells irrespective of Treg cell addition showing that our cultures had bona fide Th17 cells and Treg cells did not skew them towards induced Treg (iTreg) cell or other lineages (Fig. 1C, 1E, S2D). Supernatants derived from these cells also showed increased amounts of IL-17A and IL-21 both at early and late time points (Fig. 1F). We also examined CD45.2 Tcon and Treg cells in Th17 cell co-culture conditions. While Tcon cells were similar to Tresp cells, 30% of Treg cells lost Foxp3 expression and 5% of those cells expressed IL-17A on d3 (Fig. S3A). Control CD45.2 Treg cells that were cultured alone with IL-2 under Th17 cell polarizing conditions did not lose Foxp3 (Fig. S3A, right panel). When we followed Treg cells in Th17 co-cultures at different time points, we found that the frequency of Foxp3+ cells that started as >99%, dropped to 50% on d3, and rebounded to about 90% on d7 (Fig. S3B). Among Treg cells, we observed an initial increase in the frequency of IL-17A+Foxp3− cells (12%), which seemed unlikely to be due to outgrowth of contaminating Th17 cells since these decreased in number as Foxp3 expression was restored over the next few days. Further work will be required to verify that cells actually altered phenotype as these data imply. Taken together, these results reveal that Treg cells play one or more role(s) in promoting the induction of fully differentiated Th17 cells thereby verifying and extending the previous findings demonstrating the positive effect of Treg cells on Th17 cell differentiation (Veldhoen et al., 2006; Xu et al., 2007).

Figure 1. Up-regulation of Th17 cell associated cytokines in Tresp cells by Treg cells.

Figure 1

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A) CD45.1 Tresp cells were cultured alone or co-cultured with Foxp3−GFP−CD45.2 Tcon or Foxp3− GFP−Treg cells under Th17 cell polarizing conditions for 3 days. Intracellular cytokine staining was performed in PMA-ionomycin restimulated cells. Data from flow cytometric analyses (gated on CD45.1+ cells) shows the percentage of IL-17A+ cells (left panel) and mean fluorescence intensities (MFI) of IL-17A. (B) The frequency of IL-17A+ cells at on d3 or d4 from 5 independent experiments is depicted. Data points +/− SEM are plotted. Grey and White colored data points indicate same experiments. (C) Cells stimulated as in (B), were stained for IL-17F, IL-22, Foxp3 or IFN-γ and the data showing the percentage of respective cytokine positive cells are plotted (right panel) (** P< 0.05). Flow cytometric dot plots (gated on CD45.1+ cells) of IL-17A and Foxp3 staining (D) and IL-17F and IFN-γ (E) are shown in “x” and “y” axes respectively. (F) ELISA quantification of IL-17A (left panel) and IL-21 (right panel) in the supernatants of indicated cultures stimulated under Th17 cell polarizing conditions.

Treg cells consume IL-2 to induce IL-17A and IL-17F in Tresp cells

In the study by Veldhoen et al., the stimulatory effect of TGF-β on IL-17A production was saturated at a concentration of 0.5–1 ng/ml, whereas we used an excess of TGF-β (2 ng/ml). We also found that the increase in IL-17A by TGF-β was saturated at 1 ng/ml in our cultures (Fig. S4A). Moreover, supernatant alone from Treg cell cultures and Treg cells across a transwell dish did not induce IL-17A in Tresp cells (Fig. S4B). Treg cells enhanced IL-17A in cells that were cultured in a Th17 cell milieu both in normal media and in serum free media, therefore ruling out any effect of TGF-β in the serum (Fig. S4B, C). These data indicated that under saturating concentrations of TGF-β, Treg cells upregulated IL-17A in Tresp cells independently of TGF-β from Treg cells. Consequently, we speculated that there must be another mechanism for IL-17A enhancement by Treg cells.

IL-2 has been shown to suppress IL-17A production through a STAT-5 dependent mechanism (Laurence et al., 2007). Meanwhile, it has been previously demonstrated that Treg cells are potent consumers of IL-2 (Maloy and Powrie, 2005) and IL-2 deprivation is a fundamental aspect of their suppressive capability (Maloy and Powrie, 2005; Pandiyan and Lenardo, 2008). Therefore, we speculated that Treg cells could be increasing IL-17A production by removing the inhibitory effect of IL-2. We therefore added exogenous IL-2 to Tresp cells under Th17 cell-inducing culture conditions both in the presence or absence of Treg cells. We found that IL-17A production with or without Treg cells was drastically reduced (Fig. 2A, Fig. S4D). In parallel experiments, we observed that Il2rb−/− mice had higher amounts of IL-17A in their serum compared to wild-type (WT) mice, suggesting that a suppressive effect of IL-2 on IL-17A production can also occur in vivo (Fig S4E). These data support the general principle that IL-2 suppresses IL-17A production under physiological conditions (Laurence et al., 2007). Because we hypothesized that in the presence of excess TGF-β, IL-2 consumption by Treg cells was responsible for the enhancement of Th17 cell differentiation, we quantified IL-2 in the supernatants by ELISA. We found that IL-2 was reduced in Treg cell co-cultures compared to Tresp cell alone or Tcon cell controls consistent with our hypothesis (Fig.2B). Since we stimulated the cells with α-CD3 and α-CD28 in the absence of antigen presenting cells, IL-2 synthesis by Tresp cells was unaffected by Treg cells (Fig. 2C). These data indicate that the reduced amounts of IL-2 were most likely attributable to consumption by Treg cells rather than suppressed IL-2 production, consistent with previous reports for such culture systems (de la Rosa et al., 2004; Pandiyan et al., 2007).

Figure 2. Induction of IL-17 in Tresp cells is dependent on IL-2 consumption by Treg cells on day-3.

Figure 2

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(A) Tresp cells were stimulated under Th17 cell skewing conditions with Tcon or Treg cells as in Fig.1B. Intracellular staining of IL-17A and Foxp3 in Tresp cells with Tcon or with Treg cells that were stimulated for 3 days with or without IL-2 added at the beginning of stimulation. (B) Loss of IL-2 accumulation in Treg-Tresp Th17 cell co-cultures. ELISA quantification of IL-2 in un-stimulated culture (open bar), in Tresp alone (light grey), Tcon (striped) or in Treg cell co-cultures (black) stimulated for 4 days. Results represent the mean +/− SD. (C) The percentage of IL-2+ Tresp cells in co-cultures is shown. The results are representative of at least 3 independent experiments. (D) Flow cytometric histograms of intracellular P-STAT5 staining of WT or Il2−/− Tresp cells (left panel) showing the MFI of the P-STAT5 staining (right panel) in the indicated cultures. (E) Intracellular IL-17A staining of WT, Il2−/− Tresp cells with or without Treg cells or 100U/ml of IL-2. At least three independent experiments showed similar results.

We also looked for other molecular concomitants in Tresp cells reflecting IL-2 consumption in Treg cell co-cultures. Decreased IL-2 receptor (CD25) expression and decreased STAT-5 signaling are clear manifestations of T cells experiencing reduced IL-2 signals (Laurence et al., 2007). In our co-cultures, we found that the presence of Treg cells caused decreased CD25 expression and markedly reduced phosphorylation of STAT-5 (P-STAT5), indicating IL-2 was sufficiently reduced to cause signaling changes in Tresp cells (Fig S4F, Fig. 2D). To further examine the effects of IL-2 in our Th17 cell cultures, we stimulated Il2−/− Tresp cells with WT Tcon or Treg cells. We found that Il2−/− Tresp cells alone displayed very low P-STAT-5 amounts and was not further reduced by addition of Treg cells (Fig. 2D). On the other hand, Tresp cells cultured with WT Tcon cells induced P-STAT-5 modestly, presumably due to the IL-2 produced by the latter (Fig. 2D). More strikingly, we found that Il2−/− Tresp cells generated a high fraction of Th17 cells that was hardly affected by Treg cells (Fig. 2E). Similarly, Treg cell mediated up-regulation of IL-17F was also less prominent when cultured with Il2−/− Tresp cells (Fig S4G). Furthermore, the addition of exogenous IL-2 to the co-cultures containing Il2−/− Tresp cells, reduced the frequency of IL-17A producing cells and abrogated any inducing effect of Treg cells (Fig. 2E). Il2−/− cells did not have an enhanced ability to produce cytokines because the fraction of cells producing IFN-γ was the same as WT cells (Fig. S4G, Y axis). Hence, these data are consistent with the possibility that IL-2 consumption by Treg cells plays an important role in the up-regulation of IL-17 cytokines in Tresp cells on day 3. However, on day 6 after stimulation, additional effects of Treg cells on IL-17 production were observed (compare Fig. S4H to Fig 2E). At that later time-point, Treg cells increased IL-17A in WT and Il2−/− cells, both in the presence or absence of exogenous TGF-β (Fig. S4H, I top and middle row). This could likely be due to TGF-β secretion by Treg cells increasing IL-17A release from WT and Il2−/− cells when exogenous TGF-β is limiting (Veldhoen et al., 2006; Xu et al., 2007). We tested this conjecture by adding α-TGF-β and found that blocking TGF-β abrogated the Treg cell mediated increase in the frequency of IL-17A-producing Tresp cells (Fig. S4H, I middle and bottom row). These results show that the effect of Treg cells on Th17 cell differentiation is independent of IL-2 consumption at later time points.

Treg cell- dependent IL-2 consumption does not regulate survival in Th17 Tresp cells

To examine whether IL-2 consumption by Treg cells also regulates the survival and proliferation of Tresp cells under Th17 cell polarizing conditions, we assessed the proliferation of the Tresp cells stimulated for 4 days. As a positive control, we also stimulated Tresp cells under neutral conditions (Th0). Consistent with our previous findings (Pandiyan et al., 2007), Th0 cells showed retarded proliferation and prominent apoptosis after four days of co-culture with Treg cells, whereas Tresp cells under Th17 cell polarizing conditions proliferated extensively and did not undergo cell death (Fig. 3, A–C). Thus, Treg cells clearly do not suppress Th17 cells by inducing apoptosis at early time points as they do with other classes of CD4+ T lymphocytes. Intracellular staining of IL-2 revealed that the frequency of IL-2 producers and MFI of IL-2 were greatly increased in Th17 cells compared to Th0 cells (Fig. 3D). Thus, Treg cells were ostensibly incapable of consuming sufficient IL-2 to cause apoptosis in Th17 cells (Fig.2B). On the other hand, lower IL-2 production in Th0 cells appears to render them susceptible to IL-2 deprivation apoptosis (Pandiyan et al., 2007). Furthermore, whereas exogenous addition of IL-2 suppressed the differentiation of Th17 cells, it increased the frequency of IFN-γ producers in Th0 cells (Fig.3E). This shows that while IL-2 provides trophic sustenance and stimulates proliferation of both Th0 and Th17 cells, it apparently has opposite effects on their differentiation. This may explain why IL-2 consumption by Treg cells has differential effects on these two lineages. These findings also verify that Treg cell dependent IL-17A induction in Tresp cells is not due to altered survival or proliferation of differentiating Th17 cells. Taken together, our data reveals that IL-2 consumption by Treg cells is apparently insufficient to impact Th17 cell survival but removes an inhibitory effect on Th17 differentiation.

Figure 3. Treg cells do not induce apoptosis of Tresp cells in Th17 cell co-cultures.

Figure 3

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(A) Flow cytometric histograms of CFSE dilution of naive CD45.1 CD4 Tresp cells stimulated under non-polarizing conditions (Th0) (upper panel) or Th17 cell conditions (Th17) (lower panel) and co-cultured with CD45.2 CD4+CD25− (Tcon) cell or CD4+CD25+ (Treg) cell for 96 hours. (B) Frequency of apoptotic Tresp cells cultured for 96 hours in Th0 or Th17 cell co-cultures in the presence of Tcon (white bars) or Treg cells(black bars). (C) Absolute counts of live Tresp cells in Th17 co-cultures in the absence (white bars) or the presence (black) of Treg cells, 3 days after co-culture stimulations as in Fig 1B. Flow cytometric histograms of intracellular IL-2 staining (left panel) and MFI of IL-2 (right panel) (D), and IL-17A+ or IFN-γ+ cells (E) cultured for 96 hours in Th0 (white bars) or Th17 (black bars) conditions. In (E), 100U/ml of IL-2 was added as indicated.

Treg cells enhance Th17 cell differentiation by IL-2 consumption in vivo

We hypothesized that Treg cells may promote Th17 cell differentiation in vivo in a manner similar to what we observed in vitro, and sought to examine the effects of IL-2 and Treg cells in the B10A 5CC7 T cell receptor (TCR) transgenic (tg) model. To examine whether Treg cells regulate Th17 cell differentiation by IL-2 consumption in vivo, we transferred 6 × 105 carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled CD4+CD25− cells (Tresp cells) from congenic CD45.1 WT or Il2−/− 5CC7 Rag2−/− TCR tg mice in to B10A Rag2−/− mice and determined their IL-17A production in response to immunization with 20 µg of pigeon cytochrome C (PCC) peptide emulsified in 200 µl of complete freund’s adjuvant (CFA) (200 µg of Mycobacterium tuberculosis). Some mice received phosphate buffered saline (PBS) or 1 × 105 fresh Treg cells from donor B10A 5CC7 TCR tg mice. Four days after immunization, we found that WT 5CC7 TCR tg cells that were co-transferred with Treg cells showed increased IL-17A production in the spleen and lymph nodes (Fig. 4A). The frequency of IL-17A producers was increased in Il2−/− cells significantly above WT cells and was not affected by the addition of Treg cells (Fig. 4A, 4C, Y axis). IL-2 production was absent in Il2−/− cells, but the frequency of IL-2 producers in WT Tresp cells was not decreased by the presence of Treg cells, showing that Treg cells did not suppress IL-2 production in vivo (Fig. 4B). We also found that the frequency of IFN-γ producers was unchanged in the presence or absence of Treg cells, showing that they did not promote Th17 cell differentiation by suppressing IFN-γ in vivo (Fig. 4C, X axis). Because Treg cells did not suppress IL-2 production in WT cells and did not affect Th17 cell differentiation in Il2−/− cells, consistent with our in vitro data, we surmised that Treg cells likely enhanced IL-17 production by consuming IL-2 in vivo.

Figure 4. Induction of IL-17 in Tresp cells is dependent on IL-2 consumption by Treg cells in vivo.

Figure 4

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B10A Rag2−/− mice received 6 × 105 CFSE-labeled CD4+CD25− cells from congenic CD45.1 WT or Il2−/− 5CC7 Rag2−/−TCR tg mice. They also received PBS or 1 × 105 fresh Treg cells from donor 5CC7 TCR tg mice. 24 hr later, some mice were immunized with 20 µg of PCC peptide emulsified in 200 µl of CFA. Four days after immunization, PMA-Ionomycin stimulated cells were analyzed for the expression of cytokines. (A) The frequencies of IL-17A expressing cells in lymph nodes (LN) or spleen (SPLN) are plotted (gated on CFSE+CD4+ cells). Each data point represents data from each mouse. These data are pooled from 3 independent experiments showing similar results. Four days after immunization, cells from lymph nodes were analyzed for IL-2 (B), or IFN-γ and IL-17A (C) by flow cytometry (gated on CFSE+CD4+ cells).

Treg cells enhance Th17 cell response and the clearance of C.albicans infection in vivo

Next we sought to examine the effects of IL-2 and Treg cells in a C. albicans infection model. Oropharyngeal candidiasis is an infection by the commensal fungus C. albicans and resistance to oral C. albicans infection requires a protective Th17 cell response in mice (Conti et al., 2009). Furthermore, IFN-γ deficient mice clear the infection efficiently, showing that Th1 cells play little or no role in fungal clearance (Farah et al., 2006). C. albicans infection in mice is characterized by fungal lesions and inflammation in the tongue, decreased food intake, weight loss and eventually a moribund state. We infected WT B6 mice weighing approximately 20 grams, with C. albicans or PBS sham control (n=11) as described previously (Conti et al., 2009). We first attempted to test the effect of blocking IL-2 signals, using 0.5 mg α-CD25 (PC61 clone) or isotype control injected intraperitoneally in four mice per group. Two mice were immuno-suppressed using cortisone. We observed that even a single injection of α-CD25 rendered mice significantly more susceptible to weight loss on d3 of infection whereas uninfected mice increased in weight (Fig. S5A). We assessed the growth of C. albicans in tongue preparations and found that the mice receiving PC61 showed significantly increased fungal burden (Fig.S5B). As expected, the cortisone treated control mice, exhibited severe disease with rapid weight loss and the highest fungal burden (Fig.S5A, B). Periodic Acid Schiff (PAS) staining and histopathological examination of the tongues revealed fungal hyphae in PC61 treated mice but not in isotype control treated mice even after 5 days of infection (Fig. S5C). We also quantified IL-17A in CD4+ cells in cervical lymph nodes (CLN) from infected mice and found that PC61 significantly decreased frequency of IL-17A and IL-17F producers among CD4+ cells compared to isotype control (Fig.S5D, first two panels). By contrast, the frequency of IL-2 and IFN-γ producers was low and unaffected by either antibody (Fig. S5D, last two panels). These data implied a surprising inhibitory effect of PC61 on the resolution of C. albicans infection and IL-17 production during infection. Direct blockade of the IL-2 receptor on Th17 cells would be expected to enhance IL-17A expression and increase fungal clearance. Also, blockade of IL-2 receptor could affect proliferation of T cells or deprive nonlymphoid cells of IL-2. By contrast, PC61 administration is likely to deplete populations of CD25+cells including natural Treg cells. Removal of a fraction of Treg cells may have resulted in less buffering of IL-2, which would be consistent with the failure of Th17 cell differentiation, more severe disease, and poorer clearance of the fungus during ongoing infection. However, this inference is made cautiously because of many potential effects of blocking IL-2 on a variety of immune cell types. We therefore assessed the frequency of CD4+ Foxp3+ Treg cells and found that it was reduced by almost 50% following the single dose of PC61 antibody before infection but not with the isotype control (Fig.S5E). Interestingly, the decrease in the frequency of IL-17A producers correlated well with the decreased frequency of Foxp3+Treg cells (Fig.S5E).

The above results potentially show that depletion of Treg cells could be one factor having an adverse effect on C.albicans infection in vivo. We decided to explore this possibility more carefully. Therefore, we devised a more direct experiment in which Rag1−/− immunodeficient mice were reconstituted with naive cells with or without Treg cells followed by acute infection with C.albicans. We transferred 1.5 × 106 CD45.2 CD4+CD25−CD44− naive cells without or with 0.5 × 106 CD45.1 CD4+CD25+Treg cells. Three days after reconstitution, the mice were orally infected with C. albicans or oral sham PBS. Cortisone treated C57BL/6 mice were used as immunosuppressed controls. We assessed the cytokine production 3 days after Candida infection in vivo, and observed that Treg cells enhanced the differentiation of naïve cells in to IL-17A producing cells, whereas they did not alter IL-2 and IFN- γ production in spleen (SPLN) and cervical lymph node (CLN) (Fig. 5A, B). Correlating with higher percentage of Th17 cells, we found that mice receiving Treg cells recovered from weight loss and showed increased fungal clearance, whereas mice that did not receive them, lost weight progressively and had dramatically higher fungal burdens (Fig. 5C, D). In this adoptive transfer model, we found that naïve cells also differentiated in to IFN-γ producing cells. Although Treg cells did not impact IFN- γ production, the protective or confounding effects of IFN-γ (and Th1 cells) and potential regulatory effects of Treg cells on these cells cannot be ruled out. Therefore, we performed adoptive transfer experiments using in vitro differentiating Th17 cells (devoid of Th1 cells) in the presence or absence of Treg cells. We reconstituted CD45.2 congenic Rag1−/− mice with 4 × 105 Tresp cells and 4 × 105 Tcon or 4 × 105 Tresp cells and 4 × 105 Treg cells. These injected cells were derived from Tcon or Treg cell cultures in which CD45.1 Tresp cells were co-cultured with CD45.2 Tcon or Treg cells at a ratio of 1:1 under Th17 cell-skewing conditions for 5 days. In this model, we found that only the Candida-infected mice exhibited a significant expansion of reconstituted CD45.1+ Tresp-Th17 cells in CLN, demonstrating the fungal specific response in draining lymph nodes (Fig.S6A, bottom). Interestingly, we also observed a substantial enrichment of Foxp3+ cells among CD45.1 negative cells in CLN and tongue preparations of the Treg cell recipients compared to Tcon cell recipients suggesting that the adoptively transferred Treg cells were recruited to the site of infection (Fig.S6B, C). Consistent with the data from above experiments, we found that mice receiving Treg cells recovered from weight loss, whereas mice that received Tcon cells, lost weight progressively (Fig. 6A). Importantly, we also found that tongue sections from infected mice, reconstituted with Tresp+Tcon control cells had dramatically higher fungal burdens as compared to the mice injected with Tresp +Treg cells (Fig. 6B). On days 2 and 3 after infection, we isolated spleen (SPLN) and CLN to obtain single cell suspensions and flow cytometry analyses revealed that CD45.1 Tresp cells in Treg cell recipients contained a clearly increased population of IL-17A-producing T cells compared to Tcon cell recipients (Fig. 6C). We also found similar results when we used Tresp cells that were co-cultured with Treg cells but were separated from Treg cells before transfer into recipients (Fig. S6D). Also, tongue histological immunostaining revealed a higher percentage of RORγt+ cells infiltrating the tongue, indicating an increased Th17 cell response in the Treg cell recipients (Fig. 6D, top panel). We also detected a small fraction of Tbet+ cells (presumably Th1 cells) in the tongue, but their frequency was unaffected by the presence of Treg cells (Fig.6D, bottom panel). One of the main functions of IL-17A in host defense is to recruit neutrophils to the site of infection. Therefore we examined the neutrophil recruitment in the infected tongue, by staining for a neutrophil marker, Gr-1. We found that in the presence of Treg cells, correlating with the increased frequency of Th17 cells, there was a higher frequency of Gr-1 positive neutrophils in the tongue compared to the controls (Fig. 6E). These results clearly show that Treg cells do not suppress but decisively enhance a protective Th17 cell response that enabled mice to clear the fungus more efficiently and recover from the infection. To further show that IL-17A produced by Th17 cells is required for the recovery of mice from infection, we also injected several mice with Tresp cells that were cultured with exogenous IL-2 under Th17 cell inducing conditions. As we demonstrated above, such cells were poor IL-17A producers in vivo (data not shown), and correspondingly, we found that 2 out of 4 mice that received IL-2 treated Tresp cells succumbed to the fungus with severe weight loss and death on d3 (data not shown). This data suggests that the capacity of the transferred T cells to produce IL-17A correlates with the protection from the fungus. PAS staining of the tongue revealed extensive fungal growth in control mice that received Tresp cells only whereas mice that received Tresp+Treg cells showed almost little or no evidence of the fungus on day 5 and day 7 after infection (Fig.7). Also, mice reconstituted with Tresp cells treated with IL-2 exhibited widespread fungal growth, infiltration of cells and extensive tissue damage (Fig.7). Taken together, these results demonstrate that Treg cells could enhance immunoprotective Th17 cell responses and ameliorate infection by C. albicans.

Figure 5. Treg cells promote Th17 cell differentiation of naïve cells in vivo and protect C. albicans infected mice.

Figure 5

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Rag1−/− mice (n = 14) were reconstituted with CD4+CD25−CD44− naive cells (T Naive + PBS) without or with CD4+CD25+Treg cells (T Naive + Treg) and were infected with Candida. Some mice were infected with PBS sham controls (Uninfected + PBS). Cortisone treated C57BL/6 mice serve as immunosuppressed controls. Spleens (SPLN) or cervical lymph nodes (CLN) were harvested on day 3 after infection for IL-17A (A), IL-2 or IFN- γ (B) intracellular staining (gated on CD45.2+ CD4+ cells to exclude Treg cells except for uninfected + PBS control). (C) Percent weight change on indicated days after infection (d0–d5) is shown. (D) Tongues were harvested on day 5 after infection to assess fungal Colony Forming Units (CFU).

Figure 6. Treg cells enhance Th17 responses and protect C. albicans infected mice.

Figure 6

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(A) Rag 1−/− CD45.2 mice (n = 15) were reconstituted with Tresp+Tcon or Tresp+Treg cells that were polarized under Th17 cell conditions for 5 days. Tresp cells were obtained from CD45.1 congenic mice and Tcon and Treg cells were obtained from CD45.2 mice. Recipient mice in each group were infected with sham controls or with C. albicans. (A) The percent weight change in mice reconstituted with indicated cells and infected with C. albicans on d0. (B) Mice in indicated groups were sacrificed on day 7 after infection and tongues were harvested. (CFU)/gm of tongue tissue plated in 10 fold serial dilutions and assessed in triplicates. Mean values +/−SEM are plotted. These data are from 3 independent experiments showing similar results. Asterisks ‘*’ denote zero values for indicated groups in (B). (C) Mice were reconstituted and infected as in (A). On indicated days post-infection, cells from SPLN and CLN were re-stimulated with PMA-ionomycin to assess intracellular IL-17A (plots gated on CD45.1 Tresp cells). (D) The percentage of RORγt (top panel) and T-bet (bottom panel) positive cells in the tongue, as shown by histological immunostaining. (E) Mice were reconstituted and infected as in (A). Histological immunostaining for Gr-1 neutrophil marker in tongue on day 3 after infection (brown, denoted by arrows). Microscopic images of the slides viewed at 10X magnification. These results represent data from 3 independent experiments.

Figure 7. Treg cells enhance the clearance of C. albicans in mice.

Figure 7

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Histological evaluation of C. albicans infected mice. Mice were reconstituted with indicated cells and infected as in Fig. 6. On day 5 after infection, tongues were harvested from mice. Sections of the tongues were stained with PAS to assess inflammation and infiltration (IF) of cells and to detect C. albicans (Ca), stained pink in color. (Pa) and (Ep) denote papillae and the epithelial layer of the tongue respectively. Microscopic images of the slides viewed at 10X magnification. Results are representative of two independent experiments.

The above data raise the issue of whether during Th17 cell mediated inflammation, Treg cells remain bona fide regulatory cells that can suppress autoimmunity. We therefore carried out control experiments to test the regulatory function of Treg cells in inflammatory bowel disease (IBD) by adoptively transferring Th17 cells similar to those used in C.albicans infection model. The cells were mixtures of Thy1.1+ Tresp cells and Thy1.2+ Tcon or Thy1.2+ Treg cells, polarized under Th17 cell polarizing conditions at a ratio of 1:1 into ten congenic Thy1.2, C.B-17 scid mice. We found that the mice in Tcon and Treg cell groups started losing weight for the first 2 weeks, showing that Treg cells poorly suppressed the onset of IBD induced by differentiated Th17 cells (Fig.S7A). However, around 3 weeks, mice that received Tresp +Treg cells started regaining weight progressively and nearly matched the levels of PBS control mice, whereas the Th17 Tresp +Tcon cells continued to deteriorate (Fig.S7A). Interestingly, in Th0 and or Th1 model, in which naive cells are used for inducing IBD, Treg cells completely suppressed disease from the onset (Fig.S7B). Day 42 colonic sections revealed that mice that received Tresp with Tcon cells, had elongated crypts, massive cell infiltration, and a thickened colon wall indicating autoimmune colitis, whereas Treg cell recipients showed no sign of inflammation (Fig.S7C). These results demonstrated that the suppressive properties in Treg cells were generally intact and perhaps only slightly attenuated early in acute Th17 cell inflammation in vivo. Following co-transfer with Th17 Tresp cells, Treg cells manifested their suppressive functions over time and were capable of efficiently suppressing differentiated Th17 cells and Th17 cell-IBD in vivo. These results demonstrated that the loss of suppressive properties in Treg cells is only transient during acute Th17 cell inflammation in vivo. Treg cells resumed their suppressive functions over time and were capable of efficiently suppressing differentiated Th17 cells and Th17 cell-IBD in a delayed manner in vivo.

DISCUSSION

Although Treg cells have been previously shown to promote IL-17A induction in CD4+ cells, the overall interactions between Treg cells and Th17 cells in vitro and in vivo have remained unclear (Veldhoen et al., 2006; Xu et al., 2007). In particular, our observations on the differentiation of Th17 cells in vitro indicated that even in the presence of saturating amounts of TGF-β, Treg cells still promoted IL-17A up-regulation at early and late time points, respectively. Our data show that this most likely occurs by the high capacity of Treg cells to consume IL-2 and decrease overall IL-2 amounts in milieu of Th17 cells. This extends the observation that IL-2 consumption by Treg cells is an important component of their immunological function (de la Rosa et al., 2004; Pandiyan and Lenardo, 2008; Pandiyan et al., 2007). Our current data also indicate that IL-2 consumption by Treg cells is not necessarily suppressive but actually serves a potentially immunoprotective role in promoting Th17 cell differentiation. Previous work has shown that IL-2 inhibits IL-17A production in Th17 cells (Laurence et al., 2007), and we have verified this observation and showed that this effect, which likely influences Th17 cell differentiation, can be controlled by Treg cells. Our current finding mainly reveals that IL-2 restrains IL-17A during an early induction period, and IL-2 consumption by Treg cells may play a less prominent role at later time-points or in the maintenance phase of Th17 cell homeostasis. Interestingly, Treg− cell mediated IL-2 consumption did not cause apoptosis or retard proliferation of Th17 cells, which could be due to increased florid production of IL-2 by Th17 cells, compared to Th0 cells. This is consistent with recent quantitative models implying that apoptosis caused by Treg cell-mediated IL-2 consumption may be operational only when IL-2 amounts are limited to close proximity of the responding T cells (Busse et al., 2010; Pandiyan et al., 2007). Our findings contrast with the study of Veldhoen et al. (2006), because we found that Treg cells did not inhibit IL-2 production from Th17 cells. In their study, the sole source of TGF-β was Treg cells, which might have induced IL-17A as well as reduced IL-2 (Gunnlaugsdottir et al., 2005). In our system, Tresp cells were exposed to saturating amounts of exogenous TGF-β and α-CD28 both in the presence or absence of Treg cells and this strong co-stimulation may have prevented any Treg cell mediated inhibition of IL-2 synthesis in Tresp cells.

Consistent with the observations that Treg cells promote rather than suppress Th17 cell differentiation, we found that Treg cells potently enhanced fungal clearance and recovery from oral C. albicans infection. Thus, it is now clear that Treg cells play an important role in fighting fungal infections in addition to any effect on maintaining immunological self-tolerance or immune homeostasis. Other recent studies have shown that Treg cells confer protection against viral infections, however these mechanisms are unrelated to Th17 cell responses (Lanteri et al., 2009; Lund et al., 2008). Our findings may explain the expansion of Treg cells in response to C. albicans, resulting in reduced pathology during disseminated candidiasis infection in B7-2 deficient mice (Montagnoli et al., 2002) and why patients with Treg cell defects are prone to C. albicans infections (Coutinho and Carneiro-Sampaio, 2008; Roifman, 2000). We believe that the protective effect of Treg cells is largely mediated by an increase in IL-17A production during C. albicans infection, because IFN-γ and T-bet expression was unaffected by Treg cells. Nevertheless, IFN-γ is not essential for fungal resistance in this model (Farah et al., 2006). Taken together, these studies strongly support the notion that Treg cells may not exclusively function to “regulate” by suppressing immune responsiveness, but rather cooperate with other helper T cell subsets in immunoprotective functions against infection.

It could be speculated that Treg cell mediated suppression of inflammation contributed to the recovery of the mice from the disease. However, increased amounts of inflammatory Th17 cell cytokines and enhanced fungal clearance in the presence of Treg cells at least during early infection argue against this possibility. It is notable that we show that Treg cells maintained suppressive capacity because we found that the overall cellularity of Tresp cells was 50% lower in Treg cell recipients than controls on d7 and not on d3 after infection. Also, Treg cells suppressed at later phases in our Th17 cell-IBD model in vivo. Whether Treg cells suppress Th17 cell tissue pathology in a delayed manner, and the mechanism of this possible delayed suppression is unclear. We are currently investigating these delayed events, which may be Stat-3 dependent as indicated by a recent study (Chaudhry et al., 2009). Previous studies by us and others have implied that Treg cells forfeit suppressive capacity when overwhelming amounts of survival cytokines are available, such as during a severe, ongoing infection that involves toll-like receptor (TLR) signals (Pandiyan et al., 2007; Pasare and Medzhitov, 2003). Our current study demonstrates that Treg cells may have an important antimicrobial role in immunity especially in conjunction with Th17 cell responses. These findings open new avenues in understanding the function of this class of CD4+ T lymphocyte.

EXPERIMENTAL PROCEDURES

Mice

C57BL/6 WT or Rag1−/− (CD45.1 or CD45.2) and BALB/c (Thy1.1 and Thy1.2) mice were purchased from Jackson Laboratories. C57BL/6, B10.A, CB-17 scid mice, CD45.1 B10.A mice, WT and Il2−/− 5CC7 TCR transgenic mice in Rag2−/− background and Foxp3gfp reporter mice were purchased from Taconic farms (Germantown, NY). CB-17 scid mice were also purchased from Charles River Laboratories or from Taconic Farms (Germantown, NY). All mice were maintained at the NIAID animal facility and cared for in accordance with institutional guidelines.

Reagents and antibodies

Purified α-CD3 (145-2C11), purified α-CD28, α-CD25 (3C7), α-CD4, α-CD25, α-IL-2, α-IL-4 and α-IFN-γ were all purchased from BD Biosciences (San Diego, CA). α-CD25 (PC61), α-IL-17F, α-IL-17-A, α-TNF-α, α-Foxp3, α-CD45.1, α-CD45.2, α-Thy1.1 and α-Thy1.2 antibodies were purchased from eBiosciences (San Diego CA). TGF-β, PE conjugated IL-22 and IL-6 receptor antibodies were purchased from R&D Systems (Minneapolis, MN). Mouse CD4+ T cell isolation kit II, Anti FITC Multisort kit and α--Biotin micro-beads were purchased from Miltenyi Biotec (Auburn, CA). IL-2 and IL-17A and IL-6 Quantikine ELISA kits, recombinant mouse IL-6, IL-2 and TGF-β were purchased from R & D systems. Mouse cells were cultured in complete RPMI-1640 (Bio-Whittaker) supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 10 mM HEPES, 1 mM sodium pyruvate and 50 µM β-mercaptoethanol.

Cell purification

Splenocytes were harvested from 5 to 12 week old mice. CD4= cells were purified by negative selection using CD4+T cell isolation kit II (Miltenyi Biotec, Auburn). MACS sorted CD4+ cells were flow cytometry sorted for naïve cells i.e CD4+CD25−CD44low cells or Treg cells (>99% purity). In some experiments, we used flow cytometry sorted CD4+CD25+ GFP+ Treg cells or CD4+CD25−GFP− Tcon cells from Foxp3gfp reporter mice. The purity of CD44low CD62Lhigh CD25− naïve cells was more than 99%.

Th17 cell differentiation and co-culture with Treg cells

CD4+ CD44low CD62Lhigh CD25− naïve responder T cells (Tresp) (3×104) were co-cultured in U-bottom 96 well plates with 3×104 Tcon cells or 4×104 Treg cells in the presence of soluble 1µg/ml α-CD3 and 2µg/ml α-CD28 under Th0 or Th17 cell polarizing conditions for 3–8 days. Tresp cells were derived from congenic CD45.1 or Thy1.1 mice, and Tcon or Treg cells were derived from CD45.2 B6 or Thy1.2 BALB/C mice and so that Tresp cells could be tracked using CD45.1 or Thy1.1staining (Fig. S1). Th0 cells were stimulated only with α-CD3 and α-CD28 with no added cytokines and Th17 cells were polarized using IL-6 (20 ng/ml) TGF-β (2 ng/ml), α-IFN-γ (6 µg/ml) and α-IL-4 (6 µg/ml). The cells showed detectable IL-17A expression around d3 and started to die in the cultures around d8. Therefore, we chose d3 or 4 as early time-points and d6 or 7 as late time-points to assess cytokine production. Where indicated, Tresp cells were CFSE labeled to assess their proliferation. Cell death analyses were performed based on forward scatter or forward scatter and propidium iodide staining. When indicated, IL-2 (100 U/ml) and α-TGF-β (50 µg/ml) were added at the beginning of stimulation of co-cultures. WT or Il2−/− cells from B10.A 5CC7 TCR transgenic Rag2−/− mice were used as Tresp cells and Tcon or Treg cells were isolated from CD45.1 B10.A mice for this experiment.

IBD induction by naïve or Th17 cell transfer in vivo

For conventional IBD induction (Th0 IBD), Thy1.2, C.B-17 scid mice received 4 × 105 fresh CD45.2 CD25− CD44lowCD62LhighCD4+ congenic cells along with 4 × 105 Thy1.2 Tcon or Treg cells by intra peritoneal (IP) injection. For Th17 cell IBD, Thy1.2, C.B-17 scid mice received Th17 cells that were stimulated and differentiated for 5 days. For this, naïve Thy1.1 Tresp cells were co-cultured with fresh Tcon or Treg cells at a 1:1 ratio under Th17 cell polarizing conditions and these co-cultures were used as the source of Th17 cells. For some experiments, CD45.1 Rag1−/− mice were used as recipients and CD45.2 Tresp cells were used as donor cells. Tcon and Treg cells were derived from congenic CD45.1 mice in these experiments. The weight of the recipient mice was monitored in a blinded fashion. SPLN, MLN and the gut for isolation of lamina propria mononuclear cells (LPMC) were harvested, at indicated time-points after induction for phorbol myristate acetate (PMA)-ionomycin re-stimulation and intracellular cytokine analyses.

C. albicans infection in mice

Experiments using an oral C. albicans mouse model were performed at the University of Buffalo. All protocols were approved by SUNY Buffalo Institutional Animal Care and Use Committee. Age and sex matched C57BL/6 mice were infected and individually caged after infection, as previously described (Conti et al., 2009; Kamai et al., 2001). Briefly, they were anesthetized using a mixture of ketamine (100 mg/ml): xylazine (20 mg/ml) (2:1) solution diluted 5-fold with sterile saline. “Y” µl of anesthetic mixture was administered, Y being calculated according to weight of mouse (Y= weight of the mouse (gm) X 3.9 + 70). They were infected under anesthesia by placing a 0.0025-g cotton ball saturated with 2× 107 C. albicans (CAF2-1) blastospores sublingually for 90 min. They were treated with 225 mg/kg cortisone acetate (Sigma-Aldrich) if indicated. For experiments involving immunodeficient mice, CD45.2 Rag1−/− mice were reconstituted with Th17 cells, 3– 5 days before infection. We also performed one experiment using C.B-17 scid mice recipients and Thy1.1 Tresp donor cells and Tcon and Treg cells from Thy1.2 mice.

Histology and Intracellular staining of cytokines

For immunocytochemical hematoxylin and eosin (H&E) staining, tissues were washed with PBS, fixed with 10% formalin overnight and suspended in 70% ethanol to prevent over fixation. Paraffin sectioning and immunostaining of paraffin sections were performed by Histoserv, Inc, MD. For single-cell staining, cells were cultured as above, washed in PBS, fixed with CytoFix-Cytoperm kit (BD BioSciences). Before fixation, co-cultures were re-stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 4–6 hours, with brefeldin-A (10µg/ml) added in last 2 hours.

Flow cytometry

Data was acquired using BD FACS Calibur cytometers and were analyzed using FlowJo 8.8.4 software.

Statistical analyses

P values were calculated by Students ‘t’ test in Microsoft Excel software using unpaired, two tailed distribution and two-sample equal variance parameters or Mann-Whitney test in Prism 4.0 (GraphPad Software, Inc.).

Supplementary Material

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Acknowledgements

We thank Ron Germain, Alfred Singer, Yasmine Belkaid, Pam Schwartzberg and Andy Snow for critically reading the manuscript, Carol Trageser, Jinwoo Lee and other members of the Lenardo laboratory for valuable suggestions and help. We also thank Owen Schwartz and Lily Koo for their help in microscopy and Julie Edwards for FACS sorting. PP was supported by a fellowship from the National Research Council (NRC), National Academy of Sciences, SLG and ME were supported by NIH grant DE0188122, and SLG was also supported by AR054389. HRC was supported by a training grant (DE007034) to the Dept of Oral Biology. This work was supported by the intramural research program of National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH).

Footnotes

Author contributions. P.P. designed the study, performed experiments and analyzed data with the supervision of M.J.L. P.P and M.J.L wrote the manuscript; L.Z. measured the weight of the mice in blinded fashion and contributed to discussions; D.M helped P.P in LPMC preparations in IBD experiments. SLG and ME supported HRC. HRC performed and helped P.P in C.albicans infection experiments.

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Supplementary Materials

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