Helminths stimulate the secretion of Th2 cytokines, like IL-4, and suppress lethal graft-versus-host disease (GVHD) after bone marrow transplantation. This suppression depends on the production of immune-modulatory TGF-β and is associated with TGF-β–dependent in vivo expansion of Foxp3+ regulatory T cells (Treg). In vivo expansion of Tregs is under investigation for its potential as a therapy for GVHD. Nonetheless, the mechanism of induced and TGF-β–dependent in vivo expansion of Tregs, in a Th2 polarized environment after helminth infection, is unknown. In this study, we show that helminth-induced IL-4 production by host cells is critical to the induction and maintenance of TGF-β secretion, TGF-β–dependent expansion of Foxp3+ Tregs, and the suppression of GVHD. In mice with GVHD, the expanding donor Tregs express the Th2-driving transcription factor, GATA3, which is required for helminth-induced production of IL-4 and TGF-β. In contrast, TGF-β is not necessary for GATA3 expression by Foxp3+ Tregs or by Foxp3 CD4 T cells. Various cell types of innate or adaptive immune compartments produce high quantities of IL-4 after helminth infection. As a result, IL-4–mediated suppression of GVHD does not require invariant NKT cells of the host, a cell type known to produce IL-4 and suppress GVHD in other models. Thus, TGF-β generation, in a manner dependent on IL-4 secretion by host cells and GATA3 expression, constitutes a critical effector arm of helminthic immune modulation that promotes the in vivo expansion of Tregs and suppresses GVHD.

Allogeneic bone marrow (BM) transplantation (BMT) and hematopoietic stem cell transplantation are curative approaches for the treatment of both malignant and lethal nonmalignant disorders. The beneficial outcome of transplantation is curtailed by donor immune cell-mediated alloreactivity against host tissues, causing lethal and devastating graft-versus-host disease (GVHD) (13). Treatment options for GVHD are limited to immune-suppressive medications (i.e., steroids) that provide limited short-term and no long-term benefits and cause severe toxicity. An alternative approach to the management of lethal GVHD is the administration of donor Foxp3-positive regulatory T cells (Tregs). Administration of Tregs at adequate numbers suppresses donor cell alloreactivity and thus GVHD yet preserves the beneficial donor cell-mediated anti-tumor (graft-versus-tumor) immunity (4, 5). However, the addition of sufficient numbers of donor Tregs is a challenging and costly goal in clinical practice (6, 7), necessitating the discovery of in vivo methods to trigger immune regulatory pathways, expand functional donor Tregs, and suppress GVHD in BMT/hematopoietic stem cell transplantation patients.

Intestinal helminths have immune regulatory properties affecting the innate as well as adaptive immune pathways, and they promote the expansion of Tregs (8, 9). Helminths or helminth products can directly stimulate immune regulatory pathways of the host; for example, they can induce the expansion of Tregs (10). Several clinical trials have explored the use of helminths to suppress aberrant immunity in patients with allergic, autoimmune, or immunological disorders (11, 12). Helminths can also modulate intestinal and systemic immunity through altering the composition of commensal bacteria in mammalian gut, called microbiota (13, 14). GVHD is associated with major shifts in composition of microbiota in which the lack of specific bacterial strains is found to predispose to more severe GVHD (15, 16). Add-back of these bacterial strains suppresses intestinal inflammation and improves the outcome of BMT in mice (16). Therefore, therapeutic manipulation of the composition of intestinal microbiota, by means of fecal microbiota transplantation, synthetic stool substitutes, add-back of bacterial strains, or bacterial products, is an attractive area of basic and clinical research (12, 15, 17).

The mechanism of helminth- or microbiota-mediated immune modulation is not characterized in detail, although TGF-β appears to be a central player in helminth-induced immune suppression (18). We showed previously that TGF-β is critical to helminth-induced in vivo expansion of Tregs and helminth-induced suppression of GVHD, in a major MHC mismatch (H2b→H2d) mouse model of BMT after myeloablative conditioning regimen total body irradiation (TBI) (19). In this model, helminth infection promoted the survival of host T cells, like IL-4–producing Th2 lymphocytes, TGF-β–generating Foxp3 CD4 T cells, or Foxp3+ CD4 Tregs. Elements of the Th2 pathway of the host mitigate GVHD (2022). These include invariant NKT (iNKT) cells, a group of T lymphocytes whose Ag recognition is restricted to lipid Ags. Stimulation of host iNKT cells by cell-specific ligands or an immune regulatory conditioning regimen, called total lymphoid irradiation (TLI), promotes the expansion of Tregs and suppresses GVHD, in a manner dependent on IL-4 production by host iNKT cells (2123). Generation of IL-4 and other Th2 cytokines is driven by the transcription factor, GATA3 (24). GATA3 is also expressed by Foxp3+ Tregs, contributing to in vivo maintenance and function of Tregs (25, 26). The link between the IL-4/Th2 pathway and Treg expansion, the latter being dependent on TGF-β in helminth infection (19), is controversial; IL-4 can stimulate or inhibit Tregs (21, 2730). Moreover, Th2 and TGF-β pathways can inhibit each other (31, 32), and how both pathways remain active after helminth infection is unknown.

In this article, we report on the role of host cell Th2 cytokine IL-4 production in helminth-induced TGF-β generation and suppression of GVHD. In a model of BMT, in which we demonstrated previously that helminth-induced expansion of Tregs and suppression of GVHD depends on TGF-β (19), we show now that helminth-induced generation of TGF-β, TGF-β–dependent expansion of Tregs, and suppression of GVHD requires the production of IL-4 by host cells. Furthermore, helminth-induced production of IL-4 and TGF-β requires GATA3. With various types of immune cells stimulated to produce IL-4 after helminth infection (3336), host iNKT lymphocytes are not required as a necessary source of this cytokine. Taken together, our results demonstrate a novel link between the Th2 pathway and Treg expansion (21, 22, 37, 38), in which helminth-induced TGF-β secretion, critical to expansion of Tregs and suppression of GVHD (19), is driven by the Th2 (IL-4/GATA3) pathway.

Wild-type (WT) C57BL/6 (H2b), WT BALB/c (H2d), IL-4−/− (H2d) mice, mice that express the Cre endonuclease driven by a CD4 promoter (H2b), mice conditionally deficient for GATA3 (GATA3 flox/flox) (H2b), and mice with a T cell–specific defect in TGF-β signaling due to overexpression of a truncated TGF-β receptor II [Cd4-TGFBR2; also called TGF-β receptor II dominant negative (TGF-β RII DN)] (H2b) were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice deficient in iNKT cells (Jα18−/−; H2d) were defined before (39) and were bred at the University of Iowa. For helminth-induced immune conditioning, 5–6-wk-old male WT BALB/c, Jα18−/−, or IL-4−/− mice were inoculated with 150 Heligmosomoides polygyrus bakeri (Hpb) third-stage larvae by oral gavage. We used a modified Baermann method (40) to obtain and enrich for infective Hpb third-stage larvae (original specimens archived at the U.S. National Helminthological Collection, no. 81,930) from stool of helminth (Hpb)-infected mice. Infective larvae were stored at 4°C until used. Mice were maintained and used in accordance with the University of Iowa Animal Care and Use Committee Guidelines.

Donor BM cells were obtained from the femurs and tibias of uninfected, 5–8-wk-old male WT C57BL/6 mice. Samples were depleted of T cells using mouse panT cell beads (Dynal Biotech) according to the manufacturer’s instructions. Samples of spleen cells from uninfected, 5–8-wk-old C57BL/6 mice were magnetically enriched for donor T lymphocytes (CD3+) using a T cell isolation kit (Miltenyi Biotec).

Our studies used an acute lethal GVHD model with MHC class I and class II mismatch (19, 41). Three-week-old Hbp-infected and uninfected male WT BALB/c, Jα18−/−, or IL-4−/− recipients (H2d) were subjected to TBI using a [137Cs] source (a total of 850 cGy in two doses given 4 h apart) and were administered 10 × 106 T cell–depleted BM (TCD-BM) cells and 1.5 × 106 splenic T lymphocytes from uninfected C57BL/6 WT (H2b) donors. Mice were monitored daily for survival for up to 100 d. Disease severity was scored based on animal weight, posture, activity, fur texture, and skin integrity (4244). In parallel experiments, uninfected and Hpb-infected mice were sacrificed 6 d after BMT and subjected to analysis of cell composition by flow cytometry, grading of inflammation by histopathology, and quantitation of serum or donor T cell–produced cytokines.

To assay TGF-β cytokine secretion, CD4+ T cells were purified from splenic and mesenteric lymph nodes (MLN) of Hpb-infected and uninfected male WT BALB/c, Jα18−/−, or IL-4−/− mice, using a CD4 T Cell Isolation Kit (Miltenyi Biotec); this resulted in >98% enrichment for CD4 T cells (data not shown). To assay Hpb-mediated suppression of cytokine production by WT (C57BL/6; H2b) donor T cells during GVHD, donor CD3+ T cells from uninfected and Hpb-infected WT BALB/c, Jα18−/−, or IL-4−/− BMT recipients (hosts) (all H2d) were sorted from total splenocytes based on staining with anti-CD3 FITC and anti-H2b PE. Sorting was performed 6 d after GVHD induction, using a FACSVantage SE DiVa cell sorter (Becton Dickinson).

Six days after BMT, uninfected and Hpb-infected mice were sacrificed. The spleen and MLN were isolated for the analysis of cell composition. For surface staining, cells were suspended at 2 × 107 cells/ml in PBS with 2% FCS, and Fc receptors were blocked with a 2.4G2 mAb (Clone: 93; BioLegend). Abs for surface staining were as follows: anti-CD3 FITC, anti-CD3 PE–Cy7 (Clone: 145-2C11), anti-CD4 PE–Cy7 (Clone: GK1.5; eBioscience), anti-H2b PE, anti-H2d PE, and anti-H2b APC (Clones: SF1-1.1, SF1-1.1.1, AF6.88.5; BD Biosciences). For intracellular Foxp3 staining, the Foxp3 staining buffer and anti-Foxp3 PE, Foxp3 PE–Cy7, or Foxp3 APC Abs (Clone: FJK-16S; eBioscience) were used in accordance with the manufacturer’s instructions. For intracellular GATA3 staining, anti-GATA3 PE (Clone: L50-823; BD Biosciences) or isotype control IgG1, κ was used.

For TGF-β ELISA, MLN cells from uninfected or Hpb-infected male WT BALB/c, Jα18−/−, or IL-4−/− mice that did not undergo BMT were stimulated with anti-CD3 (Clone: 145-2C11; eBioscience) and anti-CD28 (Clone: 37.51; eBioscience) (each at 1 μg/ml) for 48 h in cell culture medium with 1% FCS and 1 mg/ml BSA (45, 46). In some experiments, purified MLN CD4 T cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 (each at 1 μg/ml) in the same medium. TGF-β cytokine concentration in acidified and realkalinized supernatants was determined using Ab pairs from R&D Systems, according to manufacturer’s instructions. Results were displayed after subtracting the TGF-β concentration in culture supernatants from TGF-β concentrations in the culture medium. IFN-γ, IL-4, and IL-10 concentrations in supernatants from parallel cultures with medium containing 10% FCS (45, 46) were analyzed using Ab pairs from R&D Systems, according to manufacturer’s instructions. To determine IL-4–mediated maintenance of TGF-β secretion, anti–IL-4 blocking (Clone: 11B11) or isotype control (rat IgG1 κ) Ab (eBioscience) were added to cultures, each at 5 μg/ml end concentration. To determine the frequency of IL-4–producing cells, splenic or MLN cell cultures were stimulated with anti-CD3/28, and brefeldin A (Thermo Fisher Scientific) was added at the last 12 h to the cultures and at 1:1000 dilution. Cells were stained for anti-CD4, Foxp3, and IL-4 APC (Clone: 11B11; Thermo Fisher Scientific). In BMT recipients, IFN-γ and TNF-α secretion was determined by ELISA from the serum or from sorted donor splenic T cell (CD3+ and H2b+) of uninfected and Hpb-infected mice 6 d after BMT, as described above. Cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 (each at 1 μg/ml) for 48 h in lymphocyte growth medium containing 10% FCS (47). Supernatants or sera were analyzed for IFN-γ and TNF-α using Ab pairs from R&D Systems.

Six days after BMT, colons and lungs from uninfected or Hpb-infected mice were fixed in 4% neutral buffered formalin and processed, and 6-μm sections were stained with H&E. Tissues were analyzed for GVHD-related inflammation, and the severity of inflammation was scored in blinded fashion by D.E. Elliot. GVHD-related colitis was graded based on the degree of inflammation and the frequency of crypt apoptosis; inflammation was graded as previously described (48) and as none (score: 0), mild (1), moderate (2), severe without ulcer (3), and severe with ulcer (4); crypt apoptosis was graded as none (score: 0), <2 per 10 crypts (1), 2–5 per 10 crypts (2), majority (>5) of crypts containing apoptotic bodies (3), and majority of crypts containing more than one apoptotic body (4). The minimal score in this grading system for colonic disease was 0 and the maximum score was 8. GVHD-related lung inflammation was graded based on the presence of perivascular cuffing, vasculitis, peribronchiolar cuffing, and alveolar hemorrhage (49). The minimal score in this grading system for lung inflammation was 0 and the maximum was 4.

Differences in survival between groups were determined by Kaplan–Meier log-rank test. Differences in cell number and composition; serum IFN-γ and TNF-α content; IFN-γ and TNF-α generation by splenic donor T cells; TGF-β, IFN-γ, IL-4, and IL-10 cytokine output of in vitro–stimulated cell cultures; and histopathological GVHD scores between groups were determined using unpaired Welch t test using the software GraphPad Prism.

Helminths that promote the expansion of Tregs and suppress GVHD stimulate the survival of Th2-polarized and IL-4–producing host cells after conditioning with TBI (19). Activated Th2 pathway can coincide with a mixed chimeric environment in states of immune tolerance after transplantation (50). Indeed, host iNKT cells also survive after conditioning with TLI, secrete IL-4, and alleviate GVHD by promoting the expansion of Foxp3+ Tregs (21, 23). Therefore, we investigated the role of host iNKT cells in helminth-induced suppression of GVHD. As TGF-β is required for helminthic suppression of GVHD and helminth-induced expansion of Tregs (19), we first investigated whether host iNKT cells are required for helminth-induced TGF-β production. We performed our analysis in Hpb-infected and uninfected iNKT−/− (Jα18−/−) mice prior to BMT. T cells are the major source of TGF-β after helminth infection (19, 45, 46). Following anti-CD3/28 stimulation, MLN cells from Jα18−/− mice colonized with the mouse nematode Hpb showed increased TGF-β secretion relative to uninfected mice (Fig. 1A). In addition, the TGF-β secretion observed in cells from Jα18−/− mice was similar to levels reported in WT strains (19, 46). Hpb infection suppressed IFN-γ and induced IL-4 as well as IL-10 secretion in MLN cells from iNKT-deficient (Jα18−/−) and WT BALB/c mice (Fig. 1B–D). These data are consistent with a requirement for TGF-β in helminthic conditioning of T cell responses (46) and demonstrate that this can occur in the absence of iNKT cells.

FIGURE 1.

Helminth-induced T cell–stimulated TGF-β production and modulation of cytokine production does not require host iNKT cells. TGF-β (A), IFN-γ (B), IL-4 (C), and IL-10 (D) concentrations in supernatants from 48-h cultures of MLN cells from Hpb-infected and uninfected 8–9-wk-old male Jα18−/− and WT BALB/c mice, as measured by ELISA. Cells were cultured in vitro with anti-CD3 and anti-CD28. Data show the mean (bar) from multiple independent experiments (scatter plots) where each dot (N) represents mean value of a single independent experiment calculated from multiple (≥3) repeats (differences between groups determined by unpaired Welch t test, p < 0.05 between uninfected and Hpb-infected as indicated for each panel).

FIGURE 1.

Helminth-induced T cell–stimulated TGF-β production and modulation of cytokine production does not require host iNKT cells. TGF-β (A), IFN-γ (B), IL-4 (C), and IL-10 (D) concentrations in supernatants from 48-h cultures of MLN cells from Hpb-infected and uninfected 8–9-wk-old male Jα18−/− and WT BALB/c mice, as measured by ELISA. Cells were cultured in vitro with anti-CD3 and anti-CD28. Data show the mean (bar) from multiple independent experiments (scatter plots) where each dot (N) represents mean value of a single independent experiment calculated from multiple (≥3) repeats (differences between groups determined by unpaired Welch t test, p < 0.05 between uninfected and Hpb-infected as indicated for each panel).

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GVHD is caused by donor T cells. Next, we tested whether Hpb infection suppressed WT donor T cell inflammatory response in Jα18−/− hosts. Inflammatory responses by WT C57BL/6 donor T cells were analyzed by in vitro IFN-γ and TNF-α secretion of purified donor T cells 6 d after BMT. Supernatants were collected from purified splenic donor T cell cultures, stimulated in vitro with plate-bound anti-CD3 and soluble anti-CD28, and assessed for IFN-γ as well as TNF-α content. Hpb infection suppressed IFN-γ and TNF-α secretion by WT donor T cells in Jα18−/− BMT mice (Fig. 2A, 2B). Similarly, serum inflammatory cytokine (IFN-γ and TNF-α) content was also suppressed in Hpb-infected Jα18−/− BMT recipients (Fig. 2C, 2D).

FIGURE 2.

Helminth-induced suppression of inflammatory cytokine generation in BMT mice does not require host iNKT cells. Concentrations of IFN-γ (A) and TNF-α (B) in supernatants of splenic donor T cell cultures isolated from uninfected or Hpb-infected iNKT-deficient; Jα18−/− or WT BALB/c BMT mice were assessed by ELISA. WT C57BL/6 donor T cells were FACS-sorted as described in 2Materials and Methods and seeded in triplicate at 105 cells per well. Wells were coated in anti-CD3, and cells were cultured with additional soluble anti-CD28. Serum concentrations of IFN-γ (C) and TNF-α (D) in uninfected and Hpb-infected animals 6 d after BMT were assessed by ELISA. Each symbol (dot) represents an independent experiment (N) and is calculated as the average of ≥3 wells from an individual mouse; bars represent the mean from multiple samples. Differences between groups determined by unpaired Welch t test, p < 0.05 between uninfected and Hpb-infected as indicated for each panel.

FIGURE 2.

Helminth-induced suppression of inflammatory cytokine generation in BMT mice does not require host iNKT cells. Concentrations of IFN-γ (A) and TNF-α (B) in supernatants of splenic donor T cell cultures isolated from uninfected or Hpb-infected iNKT-deficient; Jα18−/− or WT BALB/c BMT mice were assessed by ELISA. WT C57BL/6 donor T cells were FACS-sorted as described in 2Materials and Methods and seeded in triplicate at 105 cells per well. Wells were coated in anti-CD3, and cells were cultured with additional soluble anti-CD28. Serum concentrations of IFN-γ (C) and TNF-α (D) in uninfected and Hpb-infected animals 6 d after BMT were assessed by ELISA. Each symbol (dot) represents an independent experiment (N) and is calculated as the average of ≥3 wells from an individual mouse; bars represent the mean from multiple samples. Differences between groups determined by unpaired Welch t test, p < 0.05 between uninfected and Hpb-infected as indicated for each panel.

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Foxp3+ Tregs of recipient (host) or donor origin (4, 51, 52) suppress GVHD, and helminths promote the expansion of Tregs. Therefore, we investigated whether host iNKT cells are necessary for helminth-induced expansion of Tregs. Hpb infection resulted in an increase in Treg expansion in Jα18−/− BMT hosts of WT donor cells (Table I). Our findings suggest that helminths employ other host immune suppressive pathways besides iNKT cells to induce the expansion of Tregs. When we analyzed GVHD-related inflammation in lungs and the colons of uninfected and Hpb-infected Jα18−/− BMT mice, we observed significant suppression of inflammation by helminths in these target organs (Table II), and Hpb infection promoted survival in Jα18−/− BMT mice (Fig. 3A). Again, similar to WT BMT recipients (19), helminth infection did not alter the weight loss associated with GVHD in Jα18−/− BMT hosts (Fig. 3B). Compared to WT BALB/c BMT mice, in which helminths decreased sharply GVHD-related disease score (19), helminth-induced improvement in GVHD disease score in Jα18−/− BMT mice remained modest (Fig. 3C). Helminth-stimulated expansion of WT C57BL/6 donor Tregs in Jα18−/− BMT mice (Table I) was dependent on TGF-β because helminths did not stimulate the expansion of donor Tregs from TGF-β RII DN mice, whose T cells do not sense TGF-β because of overexpression of a truncated TGF-β receptor (Fig. 3D) (53). Based on the above parameters analyzed, our results showed that helminthic suppression of GVHD does not require host iNKT cells.

Table I.
The percentage of donor and host MLN Tregs increase in Hpb-infected Jα18−/− BMT mice
T cellFoxp3+ CD4 Treg (%, Mean ± SEM)a
Number of Foxp3+ CD4 Treg (Mean ± SEM)
Uninfected
Hpbp valueUninfectedHpbp value
Donor (n = 6)b 1.42 ± 0.11 1.99 ± 0.14 p < 0.05 2.29 ± 0.90 × 103 11.90 ± 2.93 × 103 p < 0.01 
Host (n = 6)b 8.64 ± 3.04 8.77 ± 0.97 NS 2.71 ± 0.71 × 103 46.92 ± 10.31 × 103 p < 0.01 
T cellFoxp3+ CD4 Treg (%, Mean ± SEM)a
Number of Foxp3+ CD4 Treg (Mean ± SEM)
Uninfected
Hpbp valueUninfectedHpbp value
Donor (n = 6)b 1.42 ± 0.11 1.99 ± 0.14 p < 0.05 2.29 ± 0.90 × 103 11.90 ± 2.93 × 103 p < 0.01 
Host (n = 6)b 8.64 ± 3.04 8.77 ± 0.97 NS 2.71 ± 0.71 × 103 46.92 ± 10.31 × 103 p < 0.01 

The p values as indicated between uninfected and Hpb-infected

The number of Foxp3+ MLN donor or recipient Tregs per mouse was calculated using the total number of mice used in each experiment, the total number of cells isolated from MLN cells, and the percentage of Foxp3+ CD4+ cells gated on CD3+ lymphocytes.

a

The percentage of donor Foxp3+ CD4 Tregs among all CD3+ donor T cells or the percentage of host Foxp3+ CD4 Tregs among all CD3+ host T cells are displayed.

b

The number of independent experiments.

Table II.
Hpb colonization suppresses GVHD-related inflammation in Jα18−/− and WT BALB/c BMT mice
OrganHistology Score (Mean ± SD)
Jα18−/− (Mean ± SD)
BALB/c WT (Mean ± SD)
UninfectedHpbp valueUninfectedHpbp value
Lung (n = 6)a 3.3 ± 0.8 1.8 ± 0.8 p < 0.01 3.7 ± 0.5 1.7 ± 0.5 p < 0.001 
Colon (n = 6)a 6.7 ± 0.5 4.5 ± 1.0 p < 0.01 7.2 ± 0.8 3.2 ± 1.5 p < 0.001 
OrganHistology Score (Mean ± SD)
Jα18−/− (Mean ± SD)
BALB/c WT (Mean ± SD)
UninfectedHpbp valueUninfectedHpbp value
Lung (n = 6)a 3.3 ± 0.8 1.8 ± 0.8 p < 0.01 3.7 ± 0.5 1.7 ± 0.5 p < 0.001 
Colon (n = 6)a 6.7 ± 0.5 4.5 ± 1.0 p < 0.01 7.2 ± 0.8 3.2 ± 1.5 p < 0.001 

The p values as indicated between uninfected and Hpb-infected.

a

Number of independent samples from each group: uninfected or Hpb-infected.

FIGURE 3.

Helminth-induced suppression of lethal GVHD and promotion of survival do not require host iNKT cells. (A) Kaplan–Meier survival curves for Hpb-infected or uninfected iNKT-deficient (Jα18−/−) male BMT recipients that received T cell–depleted BM (TCD-BM) cells (TCD-BM) only or TCD-BM plus total splenic T (TCD-BM plus T) cells from 5–6-wk-old male WT C57BL/6 donor mice. Cumulative data from two independent experiments. Uninfected TCD-BM only: n = 5; Hpb-infected TCD-BM only: n = 5; uninfected TCD-BM plus T: n = 6; Hpb-infected TCD-BM plus T: n = 7, p < 0.001 between uninfected TCD-BM plus T and Hpb-infected TCD-BM plus T. (B) GVHD disease score and (C) weight change of the same group of mice. Weight loss for each group of mice is displayed as percentage of weight change at different timepoints compared with initial weight. (D) Representative dot plots from MLN cells isolated 6 d after BMT, from uninfected (Uninf) or Hpb-infected Jα18−/− BMT recipients of WT C57BL/6 (WT B6) or TGF-β RII DN (DN B6) splenic T cell donors. For BMT, splenic donor T cells were obtained from uninfected mice and all groups also received donor TCD-BM (T cell–depleted BM) cells from uninfected C57BL/6 mice. MLN cells were stained for CD3, CD4, H2b, H2d, and Foxp3. Cells were gated on donor (H2b+) CD3+ T cells. Numbers represent the percentage of events in each quadrant and the percentage of Foxp3+ CD4 Tregs in the right upper quadrant. Representative example from three parallel independent experiments.

FIGURE 3.

Helminth-induced suppression of lethal GVHD and promotion of survival do not require host iNKT cells. (A) Kaplan–Meier survival curves for Hpb-infected or uninfected iNKT-deficient (Jα18−/−) male BMT recipients that received T cell–depleted BM (TCD-BM) cells (TCD-BM) only or TCD-BM plus total splenic T (TCD-BM plus T) cells from 5–6-wk-old male WT C57BL/6 donor mice. Cumulative data from two independent experiments. Uninfected TCD-BM only: n = 5; Hpb-infected TCD-BM only: n = 5; uninfected TCD-BM plus T: n = 6; Hpb-infected TCD-BM plus T: n = 7, p < 0.001 between uninfected TCD-BM plus T and Hpb-infected TCD-BM plus T. (B) GVHD disease score and (C) weight change of the same group of mice. Weight loss for each group of mice is displayed as percentage of weight change at different timepoints compared with initial weight. (D) Representative dot plots from MLN cells isolated 6 d after BMT, from uninfected (Uninf) or Hpb-infected Jα18−/− BMT recipients of WT C57BL/6 (WT B6) or TGF-β RII DN (DN B6) splenic T cell donors. For BMT, splenic donor T cells were obtained from uninfected mice and all groups also received donor TCD-BM (T cell–depleted BM) cells from uninfected C57BL/6 mice. MLN cells were stained for CD3, CD4, H2b, H2d, and Foxp3. Cells were gated on donor (H2b+) CD3+ T cells. Numbers represent the percentage of events in each quadrant and the percentage of Foxp3+ CD4 Tregs in the right upper quadrant. Representative example from three parallel independent experiments.

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As we show above, helminths do not require host iNKT cells to stimulate IL-4, TGF-β secretion, and to induce TGF-β–dependent Treg expansion. Helminths stimulate IL-4 production by various cells (3336). IL-4 production by host iNKT cells was found critical in expansion of Tregs and mitigation of GVHD (21). Although our results did not attest to a critical role of host iNKT cells in suppressing GVHD, we investigated the role of host cell IL-4 production, in general, in Hpb-mediated suppression of GVHD. We explored the production of inflammatory cytokines from WT (C57BL/6) donor T cells 6 d after BM transfer into Hpb-infected or uninfected IL-4−/− or WT BALB/c BMT hosts. Supernatants from splenic WT donor T cells stimulated in vitro with plate-bound anti-CD3 and soluble anti-CD28 were assayed for IFN-γ and TNF-α content. WT donor T cell inflammatory cytokine secretion (IFN-γ and TNF-α) isolated from IL-4−/− BMT hosts was not subject to Hpb-mediated suppression of secretion of inflammatory cytokines, although those isolated from WT BALB/c mice were (Fig. 4A, 4B), as previously reported (19). TNF-α secretion by WT donor T cells isolated from uninfected and helminth-infected IL-4−/− BMT mice was low compared with TNF-α secretion by WT donor T cells isolated from uninfected and helminth-infected WT BALB/c BMT mice (Fig. 4A, 4B). Inflammatory cytokine content was also analyzed in the sera of Hpb-infected and uninfected IL-4−/− or BALB/c WT mice 6 d after BMT (Fig. 4C, 4D). As in the case of cytokine secretion by donor T cells, the absence of IL-4 production by host cells had no effect, regardless of Hpb infection (Fig. 4C, 4D). In contrast, levels of serum Th1 inflammatory cytokines were significantly reduced in Hpb-infected WT BALB/c BMT recipients (Fig. 4C, 4D). These data demonstrate that the suppression of inflammatory cytokine output in BMT mice by helminths is dependent on IL-4 production by the host, suggesting that this cytokine is critical to helminthic conditioning of the host prior to BMT.

FIGURE 4.

Helminth-induced suppression of inflammatory cytokine generation in BMT mice requires host cell IL-4 production. Concentrations of IFN-γ (A) and TNF-α (B) in supernatants of splenic donor T cell cultures isolated from uninfected or Hpb-infected IL-4−/− or WT BALB/c BMT mice were assessed by ELISA. WT donor T cells were FACS-sorted as described in 2Materials and Methods and seeded in triplicate at 105 cells per well. Wells were coated in anti-CD3, and cells were cultured with additional soluble anti-CD28. Serum concentrations of IFN-γ (C) and TNF-α (D) in uninfected and Hpb-infected animals 6 d after BMT were assessed by ELISA. Each symbol (dot) represents an independent experiment (N) and is calculated as the average of ≥3 wells from an individual mouse; bars represent the mean from multiple samples. Differences between groups determined by unpaired Welch t test, p < 0.05 between uninfected and Hpb-infected as indicated for each panel.

FIGURE 4.

Helminth-induced suppression of inflammatory cytokine generation in BMT mice requires host cell IL-4 production. Concentrations of IFN-γ (A) and TNF-α (B) in supernatants of splenic donor T cell cultures isolated from uninfected or Hpb-infected IL-4−/− or WT BALB/c BMT mice were assessed by ELISA. WT donor T cells were FACS-sorted as described in 2Materials and Methods and seeded in triplicate at 105 cells per well. Wells were coated in anti-CD3, and cells were cultured with additional soluble anti-CD28. Serum concentrations of IFN-γ (C) and TNF-α (D) in uninfected and Hpb-infected animals 6 d after BMT were assessed by ELISA. Each symbol (dot) represents an independent experiment (N) and is calculated as the average of ≥3 wells from an individual mouse; bars represent the mean from multiple samples. Differences between groups determined by unpaired Welch t test, p < 0.05 between uninfected and Hpb-infected as indicated for each panel.

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In parallel, we harvested colons and lungs of Hpb-infected and uninfected IL-4−/− and WT BALB/c mice 6 d after BMT. Abundant mononuclear cell infiltrates and apoptotic bodies were present in the colons of Hpb-infected and uninfected IL-4−/− mice as well as uninfected WT BALB/c mice (Fig. 5). Similarly, dense infiltrates were evident in the lungs in the same groups. Unlike in WT BALB/c (19) (Fig. 5) or Jα18−/− (Table II) BMT recipients, Hpb colonization did not reduce the histopathological GVHD disease score in IL-4−/− BMT mice (Fig. 5).

FIGURE 5.

Helminths do not suppress GVHD-related end-organ damage in lung and the colon in IL-4−/− BMT mice. Histopathological analysis of lung (original magnification ×10) (A and E) and the colon (original magnification ×10) (B and F) (original magnification ×40) (C and G) from uninfected and Hpb-infected IL-4−/− (A–D) or WT BALB/c (E–H) BMT mice. Organs were harvested 6 d after BMT; tissue preparation and scoring between groups (D and H) was performed as detailed in 2Materials and Methods. Inflammation in the colon was characterized by mononuclear cell infiltrates, apoptotic cells filling crypts (black arrows), and apoptotic bodies (white arrows). Each symbol (dot) is an independent experiment (N) and represents the histopathology score from an individual mouse; bars represent the mean from multiple samples. Differences between groups determined by unpaired Welch t test, p < 0.05 between uninfected and Hpb-infected as indicated for each panel.

FIGURE 5.

Helminths do not suppress GVHD-related end-organ damage in lung and the colon in IL-4−/− BMT mice. Histopathological analysis of lung (original magnification ×10) (A and E) and the colon (original magnification ×10) (B and F) (original magnification ×40) (C and G) from uninfected and Hpb-infected IL-4−/− (A–D) or WT BALB/c (E–H) BMT mice. Organs were harvested 6 d after BMT; tissue preparation and scoring between groups (D and H) was performed as detailed in 2Materials and Methods. Inflammation in the colon was characterized by mononuclear cell infiltrates, apoptotic cells filling crypts (black arrows), and apoptotic bodies (white arrows). Each symbol (dot) is an independent experiment (N) and represents the histopathology score from an individual mouse; bars represent the mean from multiple samples. Differences between groups determined by unpaired Welch t test, p < 0.05 between uninfected and Hpb-infected as indicated for each panel.

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Next, we investigated whether helminth-induced suppression of GVHD is dependent on IL-4. Hpb-infected and uninfected IL-4−/− mice that received only TCD-BM cells showed minimal signs of disease throughout the 100-d follow-up period and survived for the duration of the experiment (Fig. 6). By contrast, Hpb-infected and uninfected IL-4−/− mice that received splenic T cells in addition to TCD-BM cells had increased disease scores, a reduction in body weight and, ultimately, all succumbed to disease 40–50 d after BMT (Fig. 6). When we repeated the BMT survival experiments in Jα18−/− and IL-4−/− hosts in parallel with WT BALB/c mice, we observed that helminthic protection from lethal GVHD was similar between Jα18−/− and WT BALB/c strains (Fig. 7A), whereas helminth-infected IL-4−/− BMT mice, unlike their WT BALB/c counterparts, were not protected from lethal GVHD (Fig. 7B). These results solidify the role of host cell IL-4 production, generated by various innate and adaptive immune cells after helminth infection, in Hpb-induced control of GVHD.

FIGURE 6.

Helminth-induced suppression of GVHD and promotion of survival are dependent on host cell IL-4 production. Upper panel, Kaplan–Meier survival curves of Hpb-infected or uninfected IL-4−/− male BMT recipients that received T cell–depleted (TCD-BM) cells (TCD BM) or TCD-BM plus total splenic T cells (TCD-BM + T) from 5–6-wk-old male WT C57BL/6 donor mice. Cumulative data from three independent experiments. N: cumulative number of BMT mice in each group; n = 10: uninfected TCD-BM; n = 10: Hpb-infected TCD-BM; n = 15: uninfected TCD-BM + T; n = 15: Hpb-infected TCD-BM + T. Middle panel, GVHD disease score; lower panel, weight change of the same group of mice. Weight loss for each group of mice is displayed as percentage of weight change at different timepoints compared with initial weight.

FIGURE 6.

Helminth-induced suppression of GVHD and promotion of survival are dependent on host cell IL-4 production. Upper panel, Kaplan–Meier survival curves of Hpb-infected or uninfected IL-4−/− male BMT recipients that received T cell–depleted (TCD-BM) cells (TCD BM) or TCD-BM plus total splenic T cells (TCD-BM + T) from 5–6-wk-old male WT C57BL/6 donor mice. Cumulative data from three independent experiments. N: cumulative number of BMT mice in each group; n = 10: uninfected TCD-BM; n = 10: Hpb-infected TCD-BM; n = 15: uninfected TCD-BM + T; n = 15: Hpb-infected TCD-BM + T. Middle panel, GVHD disease score; lower panel, weight change of the same group of mice. Weight loss for each group of mice is displayed as percentage of weight change at different timepoints compared with initial weight.

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FIGURE 7.

Helminth-induced suppression of lethal GVHD and promotion of survival do not require host iNKT cells but require IL-4 generation by host cells. (A) Kaplan–Meier survival curves for Hpb-infected or uninfected iNKT-deficient (Jα18−/−) and WT BALB/c male BMT recipients that received TCD-BM cells (TCD BM) only or TCD-BM plus total splenic T (TCD-BM + T) cells from 5–6-wk-old male WT C57BL/6 donor mice. Cumulative data from multiple independent experiments that involved Jα18−/− and BALB/c WT hosts (N: cumulative number of BMT mice from multiple experiments); *p: NS between WT C57BL/6 TCD-BM + T donor cells into Hpb-infected Jα18−/− and WT C57BL/6 TCD-BM + T donor cells into Hpb-infected BALB/c WT hosts. (B) Kaplan–Meier survival curves for Hpb-infected or uninfected IL-4−/− and WT BALB/c male BMT recipients of C57BL/6 WT donors. p < 0.001 between TCD-BM + T donor cells into Hpb-infected IL-4−/− and TCD-BM + T donor cells into Hpb-infected BALB/c WT hosts.

FIGURE 7.

Helminth-induced suppression of lethal GVHD and promotion of survival do not require host iNKT cells but require IL-4 generation by host cells. (A) Kaplan–Meier survival curves for Hpb-infected or uninfected iNKT-deficient (Jα18−/−) and WT BALB/c male BMT recipients that received TCD-BM cells (TCD BM) only or TCD-BM plus total splenic T (TCD-BM + T) cells from 5–6-wk-old male WT C57BL/6 donor mice. Cumulative data from multiple independent experiments that involved Jα18−/− and BALB/c WT hosts (N: cumulative number of BMT mice from multiple experiments); *p: NS between WT C57BL/6 TCD-BM + T donor cells into Hpb-infected Jα18−/− and WT C57BL/6 TCD-BM + T donor cells into Hpb-infected BALB/c WT hosts. (B) Kaplan–Meier survival curves for Hpb-infected or uninfected IL-4−/− and WT BALB/c male BMT recipients of C57BL/6 WT donors. p < 0.001 between TCD-BM + T donor cells into Hpb-infected IL-4−/− and TCD-BM + T donor cells into Hpb-infected BALB/c WT hosts.

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Next, we tested whether helminth-induced expansion of donor Tregs is IL-4 dependent. Relative to uninfected WT BMT recipient mice, counterparts infected with Hpb showed an increase in the percentage and total number of donor as well as host Tregs in both the spleen and MLN 6 d after BMT (Figs. 8, 9), similar to our previous results in WT BMT hosts (19). However, in uninfected IL-4−/− BMT recipients, analysis of the spleen and MLN at the 6-d timepoint revealed a sharp decrease in the percentage and number of Tregs of both origins in donor and host Tregs (Figs. 8, 9). Moreover, Hpb infection had no effect on the percentage or total number of host and donor Tregs in this context (Figs. 8, 9). These observations revealed that host IL-4 plays a broad role in the expansion of donor Tregs and emphasize the importance of Th2-mediated suppression of GVHD. Thus, infection with an intestinal helminth stimulated Th2 immunity and immune regulatory pathways.

FIGURE 8.

Helminth-induced increase in Foxp3+ Treg percentage requires host cell IL-4 production. Representative dot plots from spleen (upper rows) and MLN (lower rows) cells isolated from uninfected and Hpb-infected IL-4−/− or WT (BALB/c) mice 6 d after BMT. Spleen and MLN cells were stained for CD3, CD4, H2b, H2d, and Foxp3. Cells were gated on donor or host CD3+ T cells. Numbers represent the percentage of events in each quadrant and the percentage of Foxp3+ CD4 Tregs in the right upper quadrant.

FIGURE 8.

Helminth-induced increase in Foxp3+ Treg percentage requires host cell IL-4 production. Representative dot plots from spleen (upper rows) and MLN (lower rows) cells isolated from uninfected and Hpb-infected IL-4−/− or WT (BALB/c) mice 6 d after BMT. Spleen and MLN cells were stained for CD3, CD4, H2b, H2d, and Foxp3. Cells were gated on donor or host CD3+ T cells. Numbers represent the percentage of events in each quadrant and the percentage of Foxp3+ CD4 Tregs in the right upper quadrant.

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FIGURE 9.

Helminth-induced increase in Foxp3+ Treg percentage and number requires host cell IL-4 production. Data from multiple samples (N) were analyzed as detailed in Fig. 7 and displayed as dot-plot distribution with means (bar). Differences between groups were determined by unpaired Welch t test. Significant p values (< 0.05) as shown in each graph between groups.

FIGURE 9.

Helminth-induced increase in Foxp3+ Treg percentage and number requires host cell IL-4 production. Data from multiple samples (N) were analyzed as detailed in Fig. 7 and displayed as dot-plot distribution with means (bar). Differences between groups were determined by unpaired Welch t test. Significant p values (< 0.05) as shown in each graph between groups.

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Helminth infection induces the production of TGF-β and IL-4 (46, 47). We have demonstrated that helminthic suppression of GVHD and induction of Tregs is dependent on these two cytokines (Figs. 39 (19)). As TGF-β is the cytokine critical for helminth-induced expansion of Tregs and the suppression of GVHD, we investigated the role of IL-4 in helminthic induction of TGF-β. WT and IL-4−/− mice were colonized with Hpb, and TGF-β secretion by MLN cells following stimulation with anti-CD3/28 was assessed. In MLN cells obtained from Hpb-infected WT mice, the increase in TGF-β secretion was significantly higher than in uninfected WT cells (Fig. 10A). In contrast, TGF-β cytokine secretion was not induced in MLN cell cultures from IL-4−/−Hpb-infected mice, suggesting that IL-4 is necessary for Hpb-induced TGF-β output (Fig. 10A). Hpb infection also stimulates IL-4 and TGF-β secretion from purified T cells (19, 47). Therefore, we investigated a possible direct effect of IL-4 on TGF-β secretion in MLN T cells from Hpb-infected WT mice. Treatment of WT Hpb-infected MLN T cells with anti–IL-4 Abs reduced TGF-β secretion by ∼50% relative to cells that were treated with an isotype control Ab or cells that were left untreated. (Fig. 10B). These data suggest that IL-4 is necessary not only for the induction of TGF-β, but also for maintaining its secretion. In the absence of IL-4 and TGF-β in Hpb-infected IL-4−/− mice, helminthic suppression of Th1 and helminthic induction of IL-10 were impaired (Supplemental Fig. 1). When we explored the origin of IL-4 among CD4 T cell subsets in helminth-infected WT BALB/c and Jα18−/− mice, we observed that almost all IL-4–producing cells were Foxp3 CD4 T cells (Th2 lymphocytes), and IL-4 production from Foxp3+ CD4 Tregs was undetectable after primary infection with Hpb (Fig. 10C). We previously reported TGF-β production is evident from Foxp3+ CD4 Tregs and Foxp3 CD4 T cells (19). By contrast, we show in this article that IL-4 production was only evident in Foxp3 CD4 T cells (Fig. 10C). Thus, IL-4 generated by Foxp3 Th2 cells appears to be critical to the induction and maintenance of TGF-β production by Foxp3 CD4 T cells and Foxp3+ CD4 Tregs.

FIGURE 10.

Helminthic induction and maintenance of TGF-β production requires IL-4 production by Foxp3 CD4 T cells. (A) TGF-β concentration in supernatants of anti-CD3/28–stimulated MLN cultures from Hpb-infected and uninfected 8–9-wk-old male IL-4−/− or WT BALB/c mice, as measured by ELISA. Data show mean ± SD from ≥3 independent experiments, with each experiment containing multiple determinations (N indicates the number of independent determinations). The p value as indicated on the figure between Hpb-infected versus uninfected groups. Differences between groups determined by unpaired Welch t test. (B) Anti-CD3/28–stimulated MLN T cells from Hpb-infected WT BALB/c mice, as described in 2Materials and Methods, were cultured with anti–IL-4–blocking [anti–IL-4 (+)] isotype control Abs (Isotype Control) or no Ab added [anti–IL-4(−)], as indicated. Supernatants were analyzed for TGF-β content by ELISA. Data show mean ± SD from a representative experiment of five independent experiments, with each experiment containing multiple determinations. Differences between groups determined by unpaired Welch t test, p values as shown between groups. (C) Representative dot plots of anti-CD3/28–stimulated splenocyte and MLN cultures from Hpb-infected 8–9-wk-old male WT BALB/c or Jα18−/− mice, with brefeldin A added to cultures for the last 12 h. Cells were stained for CD4, Foxp3, and IL-4 using Foxp3 staining protocol. Data are representative examples of three independent experiments for each group.

FIGURE 10.

Helminthic induction and maintenance of TGF-β production requires IL-4 production by Foxp3 CD4 T cells. (A) TGF-β concentration in supernatants of anti-CD3/28–stimulated MLN cultures from Hpb-infected and uninfected 8–9-wk-old male IL-4−/− or WT BALB/c mice, as measured by ELISA. Data show mean ± SD from ≥3 independent experiments, with each experiment containing multiple determinations (N indicates the number of independent determinations). The p value as indicated on the figure between Hpb-infected versus uninfected groups. Differences between groups determined by unpaired Welch t test. (B) Anti-CD3/28–stimulated MLN T cells from Hpb-infected WT BALB/c mice, as described in 2Materials and Methods, were cultured with anti–IL-4–blocking [anti–IL-4 (+)] isotype control Abs (Isotype Control) or no Ab added [anti–IL-4(−)], as indicated. Supernatants were analyzed for TGF-β content by ELISA. Data show mean ± SD from a representative experiment of five independent experiments, with each experiment containing multiple determinations. Differences between groups determined by unpaired Welch t test, p values as shown between groups. (C) Representative dot plots of anti-CD3/28–stimulated splenocyte and MLN cultures from Hpb-infected 8–9-wk-old male WT BALB/c or Jα18−/− mice, with brefeldin A added to cultures for the last 12 h. Cells were stained for CD4, Foxp3, and IL-4 using Foxp3 staining protocol. Data are representative examples of three independent experiments for each group.

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GATA3 is an essential transcription factor in Th2 development, and it plays an important role in IL-4 production (24). GATA3 is also expressed in Foxp3+ Tregs and contributes to the function of these cells (25, 26). Furthermore, GATA3−/− Tregs fail to expand in the intestine after colonization with Hpb (25). Because expansion of Tregs in BMT mice after helminth infection is dependent on TGF-β, we analyzed the relationship between GATA3 expression, IL-4, and TGF-β production. WT donor Foxp3+ CD4 Tregs and Foxp3 CD4 T cells expressed GATA3 in BMT mice with or without helminth infection (Fig. 11A). In helminth-infected mice without BMT, IL-4 and TGF-β production was only evident in cultures of GATA3-sufficient, and not in cultures of GATA3-deficient, T cells (Fig. 11B). Furthermore, when we analyzed GATA protein expression in MLN T cells from uninfected and helminth-infected TGF-β RII DN mice, whose T cells do not sense TGF-β, and their C57BL/6 counterparts, we observed that GATA3 expression did not require TGF-β (Fig. 11C). Together, these results attest to a novel role of the Th2 pathway (GATA3/IL-4) in TGF-β generation and helminthic suppression of GVHD. Hence, GATA3-driven and IL-4–mediated stimulation of TGF-β production fills a gap in knowledge of the events in GVHD that occur between triggering of the Th2 pathway and the induction of Tregs.

FIGURE 11.

GATA3 is expressed on Foxp3 CD4 T cells and Foxp3+ CD4 Tregs during GVHD and is critical to IL-4 and TGF-β production by T cells. (A) Representative dot plots from spleen and MLN cells isolated from uninfected and Hpb-infected IL-4−/− (left) or WT (BALB/c) (right) BMT recipients of WT (C57BL/6) donors 6 d after BMT. Spleen and MLN cells were stained for CD3, CD4, H2b, Foxp3, and GATA3. Cells were gated on WT C57BL/6 (H2b+) donor CD3+ CD4+ T cells. Parallel splenocyte and MLN cell isolates were stained for CD3, CD4, H2b, Foxp3, and isotype Ab (instead of GATA3) (upper panels). Numbers represent the percentage of events in each quadrant and GATA3 and GATA3+ CD4 Tregs in left upper and the right upper quadrants, respectively. Representative example from three independent experiments. (B) Purified CD4 T cells from helminth-infected mice with T cell–specific deficiency for GATA3 (GATA3 fl/fl x CD4 Cre+) and from helminth-infected control GATA3-sufficient mice (GATA3 fl/fl x CD4 Cre) were stimulated plate-bound anti-CD3 and soluble anti-CD28 for 48 h. Culture supernatants were analyzed by ELISA. Data show mean (bar) from multiple independent experiments (scatter plots) where each dot (N) represents mean value of a single independent experiment calculated from multiple (≥3) repeats (differences between groups determined by unpaired Welch t test, The p values between GATA3-deficient and GATA3-sufficient groups as indicated in each panel). (C) Representative dot plots of splenocytes from uninfected (Uninf) and Hpb-infected TGF-β RII DN (DN B6) or C57BL/6 WT (WT B6) mice. Cells were stained for CD3, CD4, Foxp3, and GATA3. Cells were gated on CD3+ CD4+ T cells. Representative example from three independent experiments.

FIGURE 11.

GATA3 is expressed on Foxp3 CD4 T cells and Foxp3+ CD4 Tregs during GVHD and is critical to IL-4 and TGF-β production by T cells. (A) Representative dot plots from spleen and MLN cells isolated from uninfected and Hpb-infected IL-4−/− (left) or WT (BALB/c) (right) BMT recipients of WT (C57BL/6) donors 6 d after BMT. Spleen and MLN cells were stained for CD3, CD4, H2b, Foxp3, and GATA3. Cells were gated on WT C57BL/6 (H2b+) donor CD3+ CD4+ T cells. Parallel splenocyte and MLN cell isolates were stained for CD3, CD4, H2b, Foxp3, and isotype Ab (instead of GATA3) (upper panels). Numbers represent the percentage of events in each quadrant and GATA3 and GATA3+ CD4 Tregs in left upper and the right upper quadrants, respectively. Representative example from three independent experiments. (B) Purified CD4 T cells from helminth-infected mice with T cell–specific deficiency for GATA3 (GATA3 fl/fl x CD4 Cre+) and from helminth-infected control GATA3-sufficient mice (GATA3 fl/fl x CD4 Cre) were stimulated plate-bound anti-CD3 and soluble anti-CD28 for 48 h. Culture supernatants were analyzed by ELISA. Data show mean (bar) from multiple independent experiments (scatter plots) where each dot (N) represents mean value of a single independent experiment calculated from multiple (≥3) repeats (differences between groups determined by unpaired Welch t test, The p values between GATA3-deficient and GATA3-sufficient groups as indicated in each panel). (C) Representative dot plots of splenocytes from uninfected (Uninf) and Hpb-infected TGF-β RII DN (DN B6) or C57BL/6 WT (WT B6) mice. Cells were stained for CD3, CD4, Foxp3, and GATA3. Cells were gated on CD3+ CD4+ T cells. Representative example from three independent experiments.

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Modulation of intestinal immune pathways is critical to suppression of devastating GVHD and aberrant immune reactivity in various disorders. Intestinal colonization by helminthic parasites suppresses aberrant immunity in mice, and accumulated evidence has linked the helminth-induced Th2 pathway to suppression of inflammation (8). Nonetheless, helminth-induced suppression of aberrant immunity also requires immune regulatory cytokine TGF-β, which inhibits Th2 signaling in some in vitro or in vivo conditions (31). Moreover, TGF-β is critical for the expansion of GVHD-suppressing Tregs in helminth-colonized BMT mice (19). Although the Th2 cytokine IL-4 was shown to drive the expansion of Tregs in other models of BMT (21, 22), the link between Th2 signaling, the TGF-β pathway, and the activation of Tregs after helminth infection remains obscure.

In this article, we provide compelling evidence that helminth-induced activation of the Th2 cytokine IL-4 drives TGF-β generation and TGF-β–dependent immune suppression. We demonstrated that IL-4 production after helminth infection is driven by the Th2 transcription factor, GATA3, which is critical to the induction and maintenance of TGF-β in Hpb-infected mice. Furthermore, colonization of IL-4−/− BMT mice with Hbp fails to promote the expansion of WT donor Tregs, resulting in an inability to suppress GVHD. In contrast, Hpb infection of WT BALB/c or Jα18−/− BMT recipients triggers TGF-β–dependent expansion of WT donor Foxp3+ Tregs, which dampen lethal alloreactive responses.

Helminth infection is associated with the survival of Th2-polarized host T cells and Foxp3+ Tregs following conditioning with TBI. To understand the mechanism that links helminth-induced Th2 polarization of host cells to the TGF-β–dependent expansion of donor Tregs, we used BMT models that employ myeloablative radiation (TBI) as conditioning, because Hpb infection in this model preserves host lymphoid cells and generates a Th2-polarizing environment, similar to BMT models after conditioning by TLI (2123). BMT experiments after TLI have demonstrated that the production of IL-4 by host iNKT lymphocytes is critical for the suppression of acute GVHD (21). However, when we investigated the role of these cells in suppressing acute GVHD in Hpb-infected mice following conditioning with TBI, we observed helminth-induced suppression of GVHD in iNKT−/− (Jα18−/−) BMT recipients. These results support the notion that helminths influence immune cell subsets of the host other than iNKT in the gut and that those immune cell subsets are able to generate IL-4 and suppress acute GVHD after TBI. Hence, stimulation of host iNKT cells by TLI (21, 23) or glycolipid ligands (22) are no longer needed for helminth-induced IL-4 secretion by host cells. Our results are consistent with previous studies that showed enhanced IL-4 production by various cell types after helminth infection, in which each cell type, rather than being unique, contributed separately to optimal IL-4 production by the host (3336). These studies suggest that helminth-induced type 2 immunity and IL-4 production requires coordinated action of various cell types (54, 55).

IL-4 binds to IL-4R, whose transduction activates the transcription factor STAT6. STAT6, in turn, stimulates the expression of master regulator of the Th2 pathway, GATA3 (56). Although activation of STAT6 or GATA3 was shown to inhibit Tregs (57, 58), Tregs in Hpb-infected and Th2-polarized mice ubiquitously express GATA3 (25). Hpb infection stimulates the expansion of Tregs in inflammatory conditions like GVHD (19), and GATA3 expression by Tregs is critical to the maintenance of suppressive function of these cells in inflammation (25). Similarly, the STAT6-dependent Th2 pathway, which is essential to suppressing lethal inflammation in GVHD (22, 59), has been proposed to constitute a nonredundant signal, second to TCR, in stimulating the expansion and maintenance of peripheral Tregs (60). We propose that it is the IL-4/Th2–dependent TGF-β generation, rather than a direct effect of IL-4 or the Th2 pathway on T lymphocytes, that triggers the expansion of Tregs for the following reasons: first, the Th2 pathway is intact in T cells with TGF-β signaling defects (61). Second, helminths stimulate the Th2 pathway in T cells that are deficient in TGF-β signaling (46) and do not affect GATA3 protein expression by Foxp3+ Tregs (Fig. 11C). Third, helminths fail to promote the expansion of Tregs that do not sense TGF-β (19), although helminths promote the conversion of Foxp3+ Tregs to Foxp3 CD4 Th2 cells (62) after adoptive transfer, and this appears to be a direct effect of IL-4 on Tregs. We also propose that helminth-stimulated Th2 pathway suppresses inflammatory Th1 cells through TGF-β–dependent circuitries because helminth-triggered Th2 cells do not suppress inflammatory bowel disease or acute GVHD if TGF-β signaling to T cells is abrogated (19, 46).

The expression of Th2 transcription factor GATA3 on Tregs is known to contribute to the function of Tregs through an unknown mechanism (25, 26). We show that GATA3 is required for helminth-induced production of TGF-β by T cells, and TGF-β generated by Tregs can be essential for intestinal immune regulation (63). Although Tregs from TGF-β RII DN mice retain their ability to suppress inflammation (64), these cells do not generate TGF-β (46) but express GATA3, as we show in this study. With our data that GATA3 is critical to TGF-β generation, cellular mechanisms that lead to GATA3-dependent immune regulation by Tregs and the role of TGF-β in these immune suppressive pathways remain to be established.

In our BMT experiments, host cell IL-4 also appears to be necessary for WT donor T cell TNF-α secretion. Besides being a well-known inflammatory mediator, TNF-α activates and stimulates TGF-β generation by Tregs (65, 66). It will be interesting to know whether TNF-α plays a role in IL-4–mediated TGF-β secretion after helminth infection.

After primary infection with Hpb, cultures of splenocytes and MLN cells from WT BALB/c and Jα18−/− mice showed IL-4 production from Foxp3 CD4 T cells but not from Foxp3+ Tregs. Although Foxp3+ Tregs can be induced to generate IL-4 after secondary infection (62), our results raise the possibility that IL-4 production by Foxp3 cells drive the TGF-β production by Foxp3+ CD4 Tregs after primary helminth infection. How IL-4 signaling to Tregs contributes to GATA3 expression, TGF-β generation, and immune regulation, besides conversion to Th2 cells after adoptive transfer (62), remains to be established.

In the current study, we focused on immune conditioning of host cells by helminths. Besides cells of the host, donor T cells of helminth-infected BMT mice increase their IL-4 production (19). Several previous reports have indicated that donor Th2 cells can also alter the course of GVHD (6770). It will be important to investigate the role of the donor Th2 pathway in helminth-induced suppression of GVHD.

Although TGF-β is critical for the expansion of Tregs during the first days following BMT, alternative TGF-β–independent mechanisms of Treg expansion in a Th2-polarized environment have been reported (20). Another example implicated IL-2 signaling as a determinant of the expansion and function of Tregs (71). Experimental evidence, such as the requirement of STAT5 activation by IL-2 for early IL-4 production (72), indicates that these pathways can coordinate Th2 development, TGF-β secretion, and Treg expansion, instead of working independently from each other. Further research on these pathways can also help to understand why IL-4 inhibits Treg expansion or development in some (28, 32) and activates Tregs in other experimental settings (21, 29, 30). Similarly, in-depth exploration of these pathways (20) may explain why helminthic improvement of clinical GVHD disease score in Jα18−/− BMT recipients is modest, although helminths stimulate TGF-β–dependent regulatory pathways, suppress marked inflammation in lung as well as the colon, and promote survival in Jα18−/− BMT mice.

Collectively, our results address two important questions: First, how do Th2 and TGF-β pathways that can inhibit each other coexist after helminth infection? We answer this by showing that enhanced TGF-β generation is Th2 (GATA3/IL-4) dependent. Second, how can the Th2 pathway constitute the second signal in peripheral development and maintenance of Tregs (60)? We provide experimental evidence of a link between GATA3/IL-4, thus the Th2 pathway, and TGF-β–dependent Treg expansion in vivo. Our results deserve further attention in clinical and translational research in BMT. The Food and Drug Administration has not approved any medication for use in GVHD, and immune suppressive drugs administered to BMT recipients with GVHD do not provide clear benefit; they rather cause severe toxicity. Although the delivery of Tregs has been shown to prevent GVHD in both animal models and BMT patients, in vitro propagation or fresh isolation of enormous numbers of Tregs (6, 7) are required at a cost that is prohibitive in clinical practice. An alternative to these obstacles is the induction of Treg expansion in vivo. A more detailed understanding of mechanisms that contribute to Treg expansion in vivo, including the link between the Th2 signaling pathway and Treg expansion in helminth infection, is expected to facilitate the development of novel therapeutics for this deadly and devastating disease.

This work was supported by research funds from the National Institutes of Health: R56 AI 116715 (to M.N.I.), R01 HL56067, AI34495, HL11879 (to B.R.B.), and DK443119 (to R.S.B.), and from the Department of Veterans Affairs: BX002906 (to M.N.I.) and BX002715 (to D.E.E.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

BM

bone marrow

BMT

BM transplantation

GVHD

graft-versus-host disease

Hpb

Heligmosomoides polygyrus bakeri

iNKT

invariant NKT

MLN

mesenteric lymph node

TBI

total body irradiation

TGF-β RII DN

TGF-β receptor II dominant negative

TLI

total lymphoid irradiation

Treg

regulatory T cell

WT

wild-type.

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The authors have no financial conflicts of interest.

Supplementary data