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The Journal of Immunology, 2006, 176: 5839-5847.
Copyright © 2006 by The American Association of Immunologists

Regulatory T Cell Responses Develop in Parallel to Th Responses and Control the Magnitude and Phenotype of the Th Effector Populatio1

Justin J. Taylor*, Markus Mohrs{dagger} and Edward J. Pearce2,*

* Department of Pathobiology, University of Pennsylvania, Philadelphia, PA 19104; and {dagger} Trudeau Institute, Saranac Lake, NY 12983


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Host survival during schistosomiasis requires the development of a tightly regulated and Th2-polarized immune response against parasite egg Ags. In this system, Th1 response suppression has been thought to be enforced through the production of IL-10 by Th2 cells and natural T regulatory (Treg) cells. By comparing Th responses in schistosome egg-injected mice that lack IL-10, IL-4, and/or Treg cells, we have been able to build a detailed picture of the relative contributions of Treg cells, Th2 cells, and IL-10 to regulation of the egg-induced response. Our data indicate that eggs induce a marked Treg cell response, evident as the extensive proliferation of Foxp3+ cells that is proportionally as great as the response occurring within the Th compartment. Furthermore, we show that Treg cells prevent Th1 response development and limit the magnitude of the Th2 response. Although Treg cells are able to produce IL-10 after egg injection, we found no evidence for a role for IL-10 in Treg-mediated suppression of Th cell responses, nor did we find evidence for an inhibitory effect of Th2 cells on Th1 response development. Thus, the magnitude and phenotype of the egg-induced effector Th response are controlled by a parallel response within the Treg population.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Schistosomiasis is a chronic disease that afflicts >200 million people in developing countries (1). It is caused by infection with helminth parasites of the genus Schistosoma, and pathology is the result of the immune response to parasite eggs that have become trapped in target host tissues such as liver, intestine, or urogenital tract (2). Schistosome eggs are unusual in that they induce strongly Th2-polarized immune responses in the absence of adjuvant (3, 4, 5, 6). During infection, this Th2 response fails to clear existing parasites, but orchestrates the development of granulomas around tissue-trapped eggs and permits host survival (2, 7).

The factors underlying Th2 polarization of the egg-induced response remain unclear. In part, the Th2 bias reflects the suppressive effects of egg Ag on dendritic cell (DC) activation and the associated lack of IL-12 production by these cells during T cell priming (8). However, additional factors seem to be involved, because the available data indicate that 1) in infected IL-4–/– mice, the Th2 response is absent, and a Th1-like response emerges (9, 10); and 2) in the absence of IL-10, eggs induce Th1 and Th2 responses in which both IFN-{gamma} and IL-4, IL-5, and IL-13 are produced (11, 12, 13, 14). These findings support the view that an underlying ability of eggs to induce Th1 responses is tightly counter-regulated by Th2 cells and suggest that IL-10 is the key regulatory cytokine produced by these cells.

Recently, we and others discovered that during infection, IL-10 is made not only by Th2 cells, but also by a distinct population of CD25+ CD4 T cells that possess characteristics of regulatory T (Treg)3 cells (15, 16). CD25+ CD4 cells have been called natural Treg cells due to their existence in naive mice and their potent ability to suppress immune responses (17, 18, 19). Transcript analyses of CD25+ CD4 cells from schistosome-infected animals revealed the presence of Forkhead/winged helix transcription factor 3 (Foxp3) (16), a definitive marker for Treg cells (20, 21, 22). In adoptive transfer experiments, both Treg (CD25+ CD4) and Th2 (CD25– CD4) cells from infected wild-type (WT) mice were able to reduce the IFN-{gamma} response in schistosome-infected IL-10–/– animals (16), supporting a model in which Treg cells and Th2 cells contribute IL-10 for the suppression of Th1 responses.

In the present study we have directly examined the relative roles of Treg cells, Th2 cells, and IL-10 in the suppression of Th1 responses during the development of the polarized Th2 response against schistosome eggs. To do this, we have followed the egg-induced T cell response in the draining lymph nodes (LN) of mice injected s.c. with purified Schistosoma mansoni eggs. This system allows a distinct focus on the regulation of egg-specific Th responses in the absence of confounding and ongoing worm-specific Th responses that are present at the time (5–6 wk after infection) when egg production first begins in mice with schistosomiasis (2, 4). Our data suggest that Treg cells proliferate in response to schistosome eggs and play a pivotal role in suppressing Th1 cell development as well as limiting the magnitude of the Th2 response. The beneficial nature of such regulation is suggested by previous findings that during infection with schistosomes, both Th1 and excessive Th2 responses directed against egg Ag can have highly deleterious consequences (7, 23, 24). Strikingly, we found that although Treg cells are primed for IL-10 production, there is no evidence of a role for IL-10 in Treg-mediated effects on Th cell development or for an inhibitory effect of Th2 cells on Th1 cell development. These data allow us to propose a considerably revised model in which the immune response to schistosome eggs is characterized by the parallel activation of Th and Treg cells, and in which Treg cells, in an IL-10-independent manner, play a pivotal role in qualitatively and quantitatively molding the Th cell response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals, parasites, egg injection, and Ab depletion

C57BL/6 (B6), B6 IL-4–/–, BALB/c, and BALB/c IL-10–/– mice were purchased from The Jackson Laboratory and/or bred at University of Pennsylvania. BALB/c 4get mice were generated as described previously (25) and were maintained at University of Pennsylvania. IL-10-deficient and age-matched control mice were provided antibiotic-containing water (Bactrim; HiTech Pharmaceutical). S. mansoni (Puerto Rican strain NMRI) eggs were isolated from the livers of infected mice, resuspended at 50,000 eggs/ml in PBS (Sigma-Aldrich), and stored at –70°C until use, as previously described (26). Mice were injected with 50 µl of egg suspension/hind footpad. Soluble egg Ag (SEA) was prepared from purified eggs as described previously (26) and used at 50 mg/ml. For Treg cell depletion, 1 mg of PC61 purified by protein G affinity chromatography from hybridoma culture supernatant was injected i.p. 5–7 days before egg injection. Control mice were injected with 1 mg of rat IgG (rIgG; Sigma-Aldrich) repurified in our laboratory using protein G affinity chromatography. To assess the effectiveness of Treg cell depletion after PC61 treatment, splenocytes were stained with the 7D4 anti-CD25 (BD Pharmingen; this Ab recognizes a different epitope than that recognized by PC61) and analyzed by flow cytometry.

LN isolation and cell culture

Draining popliteal LN were harvested from egg-injected mice, and pooled peripheral nondraining LN were isolated from control mice. Single-cell suspensions were prepared, and cells were resuspended at 5 x 106 cells/ml in complete T cell medium (DMEM containing 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine (all from Mediatech), 5 x 10–5 M 2-ME (Sigma-Aldrich), and 10% FCS (HyClone)) with or without the addition of 50 µg/ml SEA and were cultured in 96-well, round-bottom plates at 37°C in 5% CO2. In some experiments, LN cells were labeled with CFSE (Molecular Probes) before culture, as previously described (27). Culture supernatants were harvested at 72 h for cytokine analysis. Bone marrow-derived DCs were generated as described previously (26). DC were cultured overnight with SEA before the addition of LN cells.

Cytokine detection and flow cytometry

Cytokine ELISA were performed using paired mAb in combination with recombinant cytokine standards (BD Pharmingen), as described previously (26). For intracellular IL-4 and IFN-{gamma} detection, cells were cultured ex vivo for 5 h with 250 ng/ml ionomycin (Sigma-Aldrich), 50 ng/ml PMA (Sigma-Aldrich), and 1 µg/ml GolgiPlug (BD Pharmingen). For Ag-specific intracellular cytokine staining, cells were cultured with SEA-pulsed DCs (10:1 LN cells:DC ratio) for 5 h with 1 µg/ml GolgiPlug. However, IL-10 was not readily detectable after a 5-h ex vivo restimulation, so for intracellular IL-10 staining, MACS-sorted CD4+ cells were incubated with SEA-pulsed DCs for 72 h, followed by 5-h incubation with PMA, ionomycin, and GolgiPlug. After a 10-min incubation with Fc block (24G2, purified from hybridoma culture supernatant), cells were incubated for 20 min with FITC-, PE-, PerCP-, PE-Cy7-, allophycocyanin-, allophycocyanin-Cy7, or biotin-conjugated Abs (BD Pharmingen) against surface markers, followed by 15-min incubations with streptavidin-Pacific Blue (Molecular Probes) when necessary. Samples were fixed with 1% formaldehyde (Sigma-Aldrich; diluted in PBS) and permeablized with 0.1% saponin (Sigma-Aldrich; diluted in PBS) before staining for intracellular proteins. Foxp3 was detected using the anti-Foxp3 kit (eBioscience) following the manufacturer’s instructions. Due to a substantial loss of fluorescence after fixation, GFP was in some cases detected simultaneously with other intracellular proteins using a purified rabbit anti-GFP Ab (Abcam), followed by FITC anti-rabbit IgG Ab (Jackson ImmunoResearch Laboratories). Samples were acquired using FACSCalibur or LSR II flow cytometers (BD Biosciences) and were analyzed with FlowJo software (Tree Star).

In vivo BrdU labeling

Mice were injected i.p. with 1 mg of BrdU (Sigma-Aldrich) on the day of egg injection. Additionally, 0.8 mg/ml BrdU was added to the drinking water, and fresh BrdU/water was provided every other day throughout the experiment. BrdU incorporation was analyzed using an APC-BrdU kit as recommended by the manufacturer (BD Pharmingen).

Statistical analysis

Student’s t test was used to calculate the significance of differences between means.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Treg cells respond to schistosome eggs

To study Treg cell responses to schistosome eggs, we injected purified eggs into the footpads of naive mice and analyzed T cells in draining LN at times thereafter. We have shown previously that this injection protocol induces a strongly polarized Th2 response that peaks by 7 days after injection (4), so our work focused on this time point after immunization. For these experiments, we used 4get mice, in which IL-4 expression is coupled to the expression of GFP; in these animals, Th2 cells are identifiable as GFP+ CD4 cells (25). To visualize Treg cells, we stained for the Treg cell-specific transcription factor Foxp3 (20, 21, 22). We found that, as expected, ~10% of CD4 cells in naive mice were Foxp3+, the majority of which were clearly CD25+ (Fig. 1A). After egg injection, the percentage of CD4 cells in the draining popliteal LN that were Foxp3+ remained at ~10%. This reflected a considerable expansion of the Foxp3+ population, because the LN CD4 population had increased in size >5-fold by this time (Fig. 1B). Foxp3+ CD4 cells in draining LN were also largely CD25+ (Fig. 1A). Consistent with previous findings that eggs induce Th2 responses, analysis of T cells from the draining popliteal LN of egg-injected 4get mice revealed that ~20% of the CD4 cells were GFP+ (Fig. 1A). This reflects a >100-fold increase in the number of cells expressing IL-4 (Fig. 1B). In nondraining LN or in the LN of naive mice, <3% of CD4 cells were GFP+ (Fig. 1A). Importantly, we observed very few CD4 cells that stained for both Foxp3 and GFP on day 7 (Fig. 1A) or at any earlier time point analyzed (data not shown), indicating that Treg and Th2 cells are distinct populations.


Figure 1
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  		FIGURE 1. Schistosome eggs simultaneously stimulate Th2 and Treg cell responses. A, Gated CD4+ cells in control LN (Naive) or draining popliteal LN (PLN) and nondraining LN (NDLN) from egg-injected 4get (BALB/c) mice were analyzed ex vivo by flow cytometry for the expression of Foxp3, CD25, and GFP as a marker for IL-4 expression. Plots from representative animals are shown. B, Total numbers of CD4+, CD4+GFP/IL-4+, and CD4+Foxp3+ cells per LN from control ({circ}) and egg-injected (•) mice were determined using the percentages measured by ex vivo flow cytometry coupled with LN cell numbers. Data points represent individual mice. *, p < 0.02, as determined by Student’s t test. C, In vivo proliferation of gated CD4+Foxp3– (top panels) and CD4+Foxp3+ (bottom panels) cells in control (Naive) animals and in the NDLN and PLN of egg-injected mice (Sm Egg) was assessed using flow cytometry to measure BrdU incorporated over the 7-day period between immunization and LN collection. Numbers on the plots reflect the percentages of cells that have incorporated BrdU. D, CD4+ cells were MACS purified from egg-injected (Egg) or control (Naive) mice and restimulated for 72 h with DCs pulsed with or without SEA. After this, cells were stimulated for 5 h with PMA and ionomycin in the presence of GolgiPlug, and intracellular IL-10 and Foxp3 expression levels were determined by flow cytometry. The numbers in bold reflect the percentages of CD4+Foxp3+ cells expressing IL-10, whereas the other numbers reflect the percentages of CD4+ cells within the various quadrants. Experiments were performed two or more times with similar results.

 
The overall increase in the number of Foxp3+ cells in the LN draining sites of egg injection indicated parallel recruitment and/or proliferation of Treg cells. To test whether proliferation contributed to the increase, we measured the incorporation of BrdU in vivo over the 7-day course of the experiment. Interestingly, >40% of the Foxp3+ population in the draining LN had incorporated BrdU (Fig. 1C), suggesting that eggs promote a strong proliferative response within the Treg population. Analysis of Foxp3-negative CD4 cells revealed that 31% of these cells had incorporated BrdU after egg injection, whereas very few cells incorporated BrdU in nondraining LN or LN from naive mice (Fig. 1C); these proliferating cells reflect the Th2 response, because 65% of the BrdU+ cells also expressed GFP (data not shown). It is evident from this experiment that background incorporation of BrdU into Foxp3+ cells in naive mice or in the nondraining LN of egg-injected animals is high (Fig. 1C) due to a high degree of turnover in the natural Treg cell population under steady-state conditions (28).

We have previously reported that CD25+ cells from infected mice produce IL-10 in response to stimulation with SEA (16). To determine whether Foxp3+ Treg cells acquire the ability to produce IL-10 in response to eggs, we restimulated sorted CD4 cells from egg-injected or naive 4get mice with SEA-pulsed DCs for 72 h and analyzed intracellular IL-10 production. Using this method, we found that 14% of Foxp3+ CD4 cells from egg-injected animals (vs 1.7% in uninjected mice) produced IL-10 after stimulation with Ag (Fig. 1D). It should be noted that a considerable percentage of the CD4 cells from egg-injected mice that made IL-10 in response to SEA were Foxp3 negative (Fig. 1D); these cells were Th2 cells, because the majority of them were also GFP positive (data not shown).

Taken together, these data indicate that schistosome eggs stimulate the simultaneous activation and proliferation of both Th2 and Treg cells.

Natural Treg cells limit the immune response to schistosome eggs

Previous work suggested that a primary role of Treg cells during schistosomiasis is to prevent Th1 cell development and thereby ensure Th2 polarization (15, 16). To investigate this issue using the egg injection model, we depleted 4get mice of natural Treg cells by injecting PC61 (anti-CD25) 5–7 days before egg injection. This protocol effectively removes 90% of the CD25+ population of CD4 cells, including the majority of Foxp3+ CD4 cells (Fig. 2A). After egg injection, increases in LN size were significantly greater in the absence of Treg cells than in their presence (data not shown), indicating that Treg control LN expansion after immunization. This increase in LN size was not the result of an outgrowth of one cell population, because all cell types examined constituted similar percentages as in Treg sufficient controls (data not shown). To determine whether Th1 responses were induced by egg injection in the absence of Treg cells, we stained CD4 cells for IFN-{gamma} after a 5-h stimulation with PMA and ionomycin ex vivo. Using this method, we found that, as expected, egg injection led to the development of very few CD4 cells capable of making IFN-{gamma}, but the percentage of cells primed to produce IFN-{gamma} was nevertheless greater in the absence of Treg cells (Fig. 2B). In light of the increased size of LN in Treg cell-depleted mice, this increased percentage of IFN-{gamma}+ CD4 cells translated into a significant increase in the total number of IFN-{gamma}+ CD4 cells in responding LN (Fig. 2B). This increase was Ag specific, because draining LN cells from egg-injected, Treg cell-depleted mice secreted significantly more IFN-{gamma} upon restimulation with SEA than did cells from egg-injected, Treg cell-sufficient controls (Fig. 2D).


Figure 2
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  		FIGURE 2. Natural Treg cells restrict egg-induced Th1 and Th2 responses in vivo. A, The effect of PC61 treatment was determined by assessing percentages of LN CD4+ cells expressing CD25 (as detected by mAb 7D4) and Foxp3 at 1 wk after injection of PC61 or rIgG; PC61 treatment resulted in a 10-fold reduction in CD25+Foxp3+CD4+ cells. B–D, Groups of six 4get (BALB/c) mice were treated with PC61 or rIgG; 1 wk later, half the mice in each group were injected with eggs. Seven days later, LN were isolated for analysis. B, LN cells from egg-injected (Egg) or control (–) 4get mice were stimulated ex vivo with PMA/ionomycin in the presence of GolgiPlug for 5 h, and CD4+ cells were stained for intracellular IFN-{gamma}. Numbers represent the percentages of CD4+ cells making IFN-{gamma} (left panels). Data from all mice in the experiment are shown in the right panel, where total numbers of CD4+IFN-{gamma}+ cells per LN were determined using the percentages measured by flow cytometry coupled with LN cell numbers. The symbol key is in the top center of figure. C, LN cells were analyzed directly ex vivo for GFP expression as a marker for IL-4 expression. Numbers represent the percentages of CD4+ cells positive for GFP (left panels). Data from all mice in the experiment are shown in the right panel, where total numbers of CD4+IL-4/GFP+ cells per LN are plotted. D, IFN-{gamma}; E, IL-4; F, IL-5; G, IL-13 (in 72-h LN culture supernatants, measured by ELISA). Cells were cultured without ({diamond}, {circ}, {triangleup}, {square}) or with ({diamondsuit}, •, {blacktriangleup}, {blacksquare}) SEA. Data points represent individual mice. *, p < 0.03; **, p < 0.01 (as determined by Student’s t test). Experiments were performed three or more times with similar results.

 
While examining IFN-{gamma} production, we also assessed Th2 responses. We found that the absence of Treg cells allowed a significantly stronger Th2 response, in which the percentage of GFP+ cells within the draining LN CD4 population almost doubled, from 18 to 32% (Fig. 2C). This reflected a significant increase in the total number of Th2 cells (Fig. 2C). This increase in the number of GFP+ cells correlated with increased Th2 cytokine secretion by LN cells in response to restimulation by SEA in vitro (Fig. 2, E–G). Thus, Treg cells not only suppress the development of Th1 cells in mice responding to egg Ag, but also limit the magnitude of the dominant Th2 response.

Th2 cells do not contribute to Th1 suppression

We previously reported that during infection, both Treg cells and Th2 cells contribute to suppression of the schistosome egg-induced Th1 response (16). However, the data in Fig. 2 show that in the absence of Treg cells, there is an increased egg-Ag-specific Th2 response that nevertheless is unable to prevent the development of a Th1 response. This finding caused us to question the role of Th2 cells in the regulation of Th1 responses. To analyze the relative contributions of Th2 cells and Treg cells in Th1 suppression, we depleted Treg cells from IL-4–/– mice (29), which are unable to generate a functional Th2 response and do not produce Th2 cytokines in response to egg injection (data not shown and see Fig. 4, A and B). Analysis of Foxp3+ cells revealed that the Treg cell response is not affected by the absence of IL-4, because the total number of Foxp3+ cells increases in response to egg injection similarly in WT and IL-4–/– LN (Fig. 3, A and B). Consistent with the view that Th2 cells do not play a major role in suppressing Th1 responses, the absence of IL-4 alone had little effect on the egg-induced IFN-{gamma} response, whether assessed by the percentage of IFN-{gamma}+ CD4 cells (Fig. 3C) or by the total number of IFN-{gamma}+ CD4 cells in the LN (Fig. 3D). However, depletion of Treg cells from IL-4–/– animals had an effect similar to that in the wild-type mice, allowing the outgrowth of a substantial population of CD4 cells that were capable of making IFN-{gamma} (Fig. 3, C and D). These data were supported by results from assays in which LN cells were restimulated with SEA, and secreted IFN-{gamma} was measured by ELISA (data not shown). Notably, as far as IFN-{gamma} production is concerned, we observed no cumulative effect of the absence of IL-4 over that obtained by the depletion of Treg cells, suggesting that Treg cells are the dominant cell type responsible for regulating Th1 cell development during the acute immune response to schistosome eggs.


Figure 4
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  		FIGURE 4. Reduced IL-10 production in the absence of Th2 responses. Groups of six B6 WT and six B6 IL-4–/– mice were injected with eggs, and 7 days later, LN cells were isolated from egg-injected mice and uninjected controls and restimulated in vitro for 72 h in the presence (•, {blacksquare}, {diamondsuit}, and {blacktriangleup}) or the absence ({circ}, {square}, {diamond}, and {triangleup}) of SEA. IL-5 (A), IL-13 (B), and IL-10 (C) were measured in supernatants using ELISA. D, At 72 h, cells were incubated with PMA, ionomycin, and GolgiPlug for an additional 5 h, and intracellular IL-10 was measured in gated CD4+ cells. Numbers represent the percentages of CD4 cells that stained positively for IL-10. Data points represent individual mice. Experiments were repeated three or more times with similar results.

 

Figure 3
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  		FIGURE 3. Th2 cells do not participate in the suppression of the egg-induced Th1 response. A, Groups of three B6 WT or B6 IL-4–/– mice were injected with eggs (Egg) or left uninjected (–). Seven days later, LN were removed, and CD4 cells were analyzed for Foxp3 expression. Numbers represent percentages of CD4+ cells that are Foxp3+. B, Total numbers of CD4+ Foxp3+ cells per LN from control ({circ}, {square}, {diamond}, and {triangleup}) and egg-injected (•, {blacksquare}, {diamondsuit}, and {blacktriangleup}) mice were calculated. C, Groups of six PC61-treated or six rIgG-treated (control) WT and IL-4–/– mice were split into two groups; mice in one group were injected with eggs (Egg), whereas those in the other remained uninjected (–). Seven days later, LN cells were isolated and stimulated ex vivo for 5 h with PMA/ionomycin in the presence of GolgiPlug, and cells were analyzed by flow cytometry for intracellular IFN-{gamma}. Numbers in boxes represent the percentages of CD4+ cells that make this cytokine. Data from all mice in the experiment are shown in D, where total numbers of CD4+IFN-{gamma}+ cells per LN are plotted. Data points represent individual mice. *, p < 0.03; **, p < 0.01 (as determined by Student’s t test). The experiment was repeated three times with similar results.

 
Suppression of Th responses is IL-10 independent

IL-10 has been implicated as a critical mediator of Th1 response suppression during schistosomiasis (11, 12, 13, 14, 15, 16). However, the loss of a major IL-10-producing cell population, Th2 cells, did not result in an increase in the Th1 response (Fig. 3). This suggested either that residual Th2 cells or another IL-10-producing cell population, such as Tregs, are present in IL-4–/– mice and are sufficient to suppress Th1 responses in this system. To address the former, we measured IL-5 and IL-10 in supernatants of Ag-restimulated LN cultures and found that cells from egg-injected IL-4–/– mice made barely detectable levels of IL-5 or IL-13 (Fig. 4, A and B), suggesting the absence of Th2 cells, but did make IL-10 in response to SEA, albeit at low levels compared with those produced by cells from egg-injected WT mice (Fig. 4C). These data were reflected in single-cell analyses, where intracellular staining (ICS) revealed the persistence of a small population of egg-induced CD4 cells capable of making IL-10 even when IL-4 is absent (Fig. 4D). This population of IL-10+ CD4 cells was largely comprised of Foxp3+ Treg cells (data not shown). Furthermore, the fraction of Foxp3+ cells capable of producing IL-10 in response to egg injection was not affected by the absence of IL-4 (data not shown).

Because Treg cell depletion in IL-4–/– mice allowed accentuated egg-induced Th1 responses (Fig. 3), it was possible that IL-10 from Treg cells alone was sufficient to suppress development of the Th1 response in Th2 response-defective, IL-4–/– mice. To directly examine the role of IL-10 in determining the outcome of the Th response, we injected eggs into IL-10–/– animals. Analysis of Foxp3+ cells revealed that Treg cells are present in IL-10–/– mice, and that expansion of the Treg population in response to egg injection occurs similarly in the absence or the presence of IL-10 (Fig. 5, A and B). Surprisingly, the absence of IL-10 alone had no effect on the percentages or absolute numbers of IL-4+ or IFN-{gamma}+ CD4 cells, as measured by intracellular staining after stimulation with PMA and ionomycin (Fig. 5, C, D, F, and G) or with SEA-pulsed DCs to measure Ag-specific cytokine production (Fig. 5, E and H). The lack of effect of IL-10 on numbers of Th1 or Th2 cells in egg-injected mice coupled with the increased numbers of both these cell types in Treg cell-depleted mice indicated that Treg cell-mediated effects on Th cell responses might be IL-10 independent. This was confirmed when depletion of Treg cells before injection of eggs into IL-10–/– mice resulted in considerably enhanced Th1 and Th2 responses (Fig. 5, C–H). The effect of Treg depletion on egg-injected IL-10–/– mice was similar to that in WT animals (Fig. 5).


Figure 5
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  		FIGURE 5. Treg cells suppress egg-induced Th responses independently of IL-10. Groups of three BALB/c WT or three BALB/c IL-10–/– mice were injected with eggs (Egg) or left uninjected (–). Seven days later, LN were removed, and CD4 cells were analyzed for Foxp3 expression (A). Numbers represent percentages of CD4+ cells that are Foxp3+. B, Total numbers of CD4+ Foxp3+ cells per LN from control ({circ}, {square}, {diamond}, and {triangleup}) and egg-injected (•, {blacksquare}, {diamondsuit}, and {blacktriangleup}) mice were determined using the percentages measured by ex vivo flow cytometry coupled with LN cell numbers. C–H, Groups of six PC61-treated or six rIgG-treated control WT and IL-10–/– mice were split into two groups; half the animals were injected with eggs (Egg), and half remained uninjected (–). Seven days later, LN cells were isolated and stimulated ex vivo for 5 h with PMA/ionomycin in the presence of GolgiPlug and stained for CD4 and intracellular IL-4 (C) or IFN-{gamma} (F). Gated CD4 cells are shown. Numbers in boxes reflect the percentages of CD4+ cells that were cytokine positive. Total numbers of CD4+IL-4+ (D) and CD4+IFN-{gamma}+ (G) cells per LN were calculated using the percentages determined by flow cytometry coupled with LN cell numbers. Additionally, cells were stimulated ex vivo for 5 h with SEA-pulsed DCs in the presence of GolgiPlug and stained for CD4 and intracellular IL-4 or IFN-{gamma}; total numbers of IL-4+CD4+ and IFN-{gamma}+CD4+ cells per LN are shown in E and H, respectively. Data points represent individual mice. *, p < 0.05; **, p < 0.01 (as determined by Student’s t test). The experiments were repeated three or more times with similar results.

 
Based on these findings, we conclude that the absence of IL-10 has little effect on the outgrowth of egg Ag-specific Th1 cells in vivo after immunization with eggs. Rather, Treg cells, working in an IL-10-independent fashion, play the primary role in restricting any incipient ability of eggs to drive Th1 response development. Likewise, the modulation of Th2 responses by Treg cells is IL-10 independent.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Schistosome infection is notable for its ability to induce Th2-polarized immune responses. The magnitude of this response is illustrated clearly in this study through the use, for the first time in this system, of 4get mice, which revealed that ~20% of CD4 cells within LN draining sites of schistosome egg deposition are expressing the IL-4 gene. This immunological outcome is important, because in the absence of a Th2 response, alternative macrophage activation fails to occur, and what is normally a chronic infection turns into an acutely lethal disease (9, 10, 14, 30, 31). Previous findings indicated that polarization of the response is at least in part due to the suppression of Th1 cell outgrowth by IL-10 produced by Th2 cells, natural Treg cells, and macrophages/DCs (11, 14, 15, 16, 32, 33). In attempting to more clearly delineate the roles of IL-10 and these various cell types in the regulation of Th1 cell development, we have found that in mice injected with schistosome eggs, the major suppressor of Th1 cell development is the Treg cell, and control occurs in an IL-10-independent manner. Our results do not support a role for Th2 cells in counter-regulating Th1 cell development in this system. Importantly, the data indicate that proportionally, the proliferative response induced by schistosome egg injection within the Treg population is at least as great as that in the Th cell compartment, and Treg cells not only suppress the Th1 response, but also effectively restrain the developing Th2 response.

The increase in Th1 and Th2 responses evident when Treg cells are depleted indicates that Treg cells broadly suppress Th responses after egg injection. This is consistent with the observed increases in allergic as well as cell-mediated autoimmune disease in patients with the immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome, which is caused by mutations in Foxp3 (34). Nevertheless, there are several possibilities as to why Th2, rather than Th1, populations should expand more effectively than Th1 cells in the face of Treg-mediated suppression in LN draining sites of egg deposition. First, unlike Th1 cells, Th2 cells make IL-6, a cytokine that has been reported to alleviate suppressive effects and allow the development of effector responses (35). Second, egg Ag do not activate DC to produce IL-12 or IL-6 (26, 36), so conditions are not conducive for Th1 response establishment.

The finding that BrdU-positive Treg cells accumulate in LN draining sites of egg injection is consistent with the view that parallel homing and activation of Ag-specific Treg cells in Ag-draining LN early during the response are essential for control of effector responses (37). The BrdU data and the finding that Treg cells from egg-injected mice can produce IL-10 in response to SEA presented by DC suggest that Treg cells are capable of recognizing schistosome Ag. This is an intriguing finding, because it has been suggested that Treg, which have a polyclonal T cell repertoire that nevertheless shows only partial overlap with Th cells, recognize a broad array of self, rather than foreign, Ag (38). Although it is conceivable that in our system Treg cells are responding to self-Ag processed from host cells damaged by egg-induced inflammation or to self-Ag expressed in DC in response to exposure to SEA, it seems more likely that the cells are, in fact, responding to parasite Ag. There is good evidence for pathogen Ag-specific Treg cell activation in other infectious disease settings (19), and parallel Treg responses may be a component of most effector responses.

An unexpected finding from these studies is that the egg-specific Th2 response plays no measurable role in suppressing Th1 responses. Despite the absence of a detectable Th2 response in egg-injected, IL-4–/– mice, we found no evidence in our experiments for the outgrowth of IFN-{gamma}+ CD4 cells, nor could we detect significantly increased levels of IFN-{gamma} in the supernatants of Ag-restimulated draining LN. This situation is quite distinct from that occurring in other helminth disease models, such as Trichuris muris infection in the B6 mouse, where the absence of IL-4 results in the development of strong and easily detectable Th1 responses (39). Previous reports have provided conflicting data about IFN-{gamma} production in schistosome egg-injected or schistosome-infected IL-4–/– mice. Our results in this study are consistent with our recent findings (40) and fit best with those of Weinstock et al. (41), who reported that in the absence of IL-4, infected mice did not switch to making a Th1 response. However, it does seem clear that there is the potential for increased IFN-{gamma} production in egg-immunized or infected IL-4–/– mice (9, 10, 14, 42), but it is likely that, as might be the case in IL-10–/– mice, the cellular source of this cytokine is a non-CD4 cell. Regardless, we have found that appreciable numbers of IFN-{gamma}+ CD4 cells and significant amounts of IFN-{gamma} production by Ag-restimulated draining LN cells were evident only when mice, either WT or IL-4–/–, were depleted of Treg cells before egg injection. In this setting, we found no evidence for synergy between Treg cells and Th2 cells in suppressing development of the Th1 response. Additionally, although recent evidence has suggested that IL-4 may influence the proliferation or generation of Treg cells (43, 44), Treg expansion and function did not appear to be altered by the absence of IL-4, because we found normal numbers of Foxp3+ cells in IL-4–/– animals, and Th1 responses in egg-injected WT and IL-4–/– mice were equally affected by the depleted of Treg cells. Thus, Treg cells play a dominant role in suppressing the emergence of Th1 cells during the response to schistosome egg Ag.

A considerable body of evidence has suggested that IL-10 plays the major role in suppressing Th1 cell development in response to schistosome eggs (11, 12, 13, 14, 15, 16, 32). In the absence of IL-10, more IFN-{gamma} mRNA can be detected in diseased tissues of infected mice, and more IFN-{gamma} is secreted into the culture supernatant when spleen or mesenteric LN cells from infected mice are restimulated in vitro with Ag (13, 16, 32). Because IL-10 is a major cytokine product of Th2 as well as Treg cells, we were surprised when Treg-depleted egg-injected mice were found to have increased Th1 responses in the face of exaggerated Th2 responses (Fig. 2). This led us to examine in greater detail the role of IL-10 in Th2 response polarization after egg injection. Using IL-10–/– mice and ICS to examine cytokine production by CD4 cells after a brief stimulation ex vivo, we found similar numbers of IFN-{gamma}+ cells in egg-injected WT and IL-10–/– mice. The depletion of Treg cells from IL-10–/– mice before egg injection resulted in subsequently stronger Th1 responses, as was the case in similarly treated WT mice, indicating that the principal suppressor of Th1 response development after exposure to eggs is the Treg cell, and suppression is mediated by an IL-10-independent mechanism. Nevertheless, Treg cells induced by egg injection are capable of making IL-10, and we assume that this cytokine, made by these cells, does play important roles, most likely in suppressing pathological inflammatory reactions mediated by innate cells (45). IL-10 has been identified as a critical factor in Treg cell-mediated suppression of the IFN-{gamma}-dependent inflammation caused by other pathogens, such as Leishmania major and Helicobacter hepaticus (46, 47).

To directly compare our findings on the effect of IL-10, in general, on egg-induced Th responses with those reported previously, we cultured draining LN cells from egg-injected WT or IL-10–/– mice for 72 h in the presence or the absence of SEA and examined IFN-{gamma} levels in culture supernatants. In marked contrast to the findings of ICS, the ELISA indicated that in the absence of IL-10, Ag-restimulated LN cells made significantly more IFN-{gamma} (data not shown). The difference can be accounted for by the finding that the absence of IL-10 allows greater Ag-driven CD4 cell proliferation during the 72 h of culture required to generate supernatants for ELISA, and the final concentration of IFN-{gamma} is in great part a reflection of the number of Th1 cells in the well (data not shown). This conclusion is consistent with the results of a previous study in which splenocytes from infected WT mice, restimulated with SEA in the presence of a neutralizing anti-IL-10 Ab, were found to produce significantly more IFN-{gamma} than those in cultures without blocking Ab (11). Therefore, we conclude that the existing view that IL-10 prevents the outgrowth of egg Ag-specific Th1 cells in schistosome-infected animals could be in part erroneous and based on an in vitro peculiarity of the system. In light of these findings, we assume that elevated IFN-{gamma} transcript levels in egg-injected, IL-10–/– mice in part reflect increased IFN-{gamma} production by non-CD4 cells; a candidate for such a cell type is the NK cell (48, 49). Elucidating the exact role of IL-10, regardless of source, during infection will be important, because its absence leads to significantly more severe disease (13, 14, 32).

Based on our data, we conclude that IL-10-independent attributes of Treg cells must allow them to directly regulate Th cell responses. The ability of Treg cells to suppress T cell proliferation in vitro (50, 51) and to inhibit other immune responses, such as those that mediate sterilizing immunity against the parasitic helminth Litosomoides sigmodontis (52), are IL-10 independent, indicating that Treg cells can exert regulatory effects through multiple mechanisms. TGF-beta and CTLA-4 have been implicated in the IL-10-independent suppressive effects mediated by Treg cells (37). TGF-beta seems an unlikely mediator of Treg suppression during schistosomiasis, because studies probing the role of TGF-beta suggest that Treg suppression is intact (53). To date, the role of CTLA-4 in schistosomiasis has not been investigated in detail, primarily due to the fact that CTLA-4–/– mice die soon after birth (54, 55).

Treg cell-mediated regulation of Th2 responses may be of particular importance during schistosomiasis due to the chronic exposure to egg Ag that occurs in the infected host. S. mansoni is not killed by the immune response, and egg deposition continues for the life of the worm, which is measured in years (2). It might be expected that this situation would lead to continuous boosting of the Th2 response. It has been recognized for some time, however, that as the infection passes into the chronic phase (which in this disease in the mouse is considered to occur beyond wk 12 of infection), the magnitude of the immune response to the parasite eggs diminishes (13, 32, 56, 57, 58). This down-modulation is measurable as a decrease in Th2 cytokine production as well as in a decrease in the size of existing and newly forming granulomas (13, 32, 56, 57, 58). The mechanisms underlying the regulation of the Th2 response are not fully understood. A recent paper reported that the percentage of CD25+CD4+ cells and the overall levels of Foxp3 mRNA increase significantly during the chronic phase of infection (59), suggesting that an accumulation of Treg cells could account for the modulation of the Th2 response. Our finding that cells that are Foxp3+ have proliferated in response to schistosome eggs provides a mechanism for expansion of the Treg cell population during infection. Future studies will be aimed at directly examining whether Treg cells are responsible for modulation of immune responses during chronic schistosomiasis.


   Acknowledgments
 
We are grateful to David Artis, Colleen Kane, Jay Farrell, and Erika Pearce for suggestions and critical reading of the manuscript, and to Euihye Jung, Jason Correnti, and Ken Thompson for expert technical assistance.


   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 Institutes of Health Grant AI32573 (to E.J.P.). Schistosome life stages were provided by National Institute of Allergy and Infectious Diseases Contract N0155270. E.J.P. is a Burroughs Wellcome Fund Scholar in Molecular Parasitology. Back

2 Address correspondence and reprint requests to Dr. Edward J. Pearce, Department of Pathobiology, Room 202C Rosenthal Building, School of Veterinary Medicine, 3800 Spruce Street, Philadelphia, PA 19104-6008. E-mail address: ejpearce{at}mail.med.upenn.edu Back

3 Abbreviations used in this paper: Treg, regulatory T; DC, dendritic cell; Foxp3, Forkhead/winged helix transcription factor; LN, lymph node; rIg, rat Ig; SEA, soluble egg Ag; WT, wild type; ICS, intracellular staining. Back

Received for publication October 4, 2005. Accepted for publication February 9, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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J. Mariotti, J. Foley, U. Jung, T. Borenstein, N. Kantardzic, S. Han, J. T. Hanson, E. Wong, N. Buxhoeveden, J. B. Trepel, et al.
Ex Vivo Rapamycin Generates Apoptosis-Resistant Donor Th2 Cells That Persist In Vivo and Prevent Hemopoietic Stem Cell Graft Rejection
J. Immunol., January 1, 2008; 180(1): 89 - 105.
[Abstract] [Full Text] [PDF]