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. Author manuscript; available in PMC: 2016 Jan 31.
Published in final edited form as: J Immunol. 2014 Dec 19;194(3):1011–1020. doi: 10.4049/jimmunol.1303099

Intestinal Helminths Regulate Lethal Acute Graft Versus Host Disease and Preserve Graft Versus Tumor Effect in Mice

Yue Li *, Hung-lin Chen *, Nadine Bannick †, Michael Henry †,‡, Adrian N Holm *, Ahmed Metwali *, Joseph F Urban Jr , Paul B Rothman *,||, George J Weiner *,‡, Bruce R Blazar §, David E Elliott *, M Nedim Ince *,‡
PMCID: PMC4297687  NIHMSID: NIHMS644178  PMID: 25527786

Abstract

Donor T lymphocyte transfer with hematopoietic stem cells suppresses residual tumor growth (graft-versus-tumor; GVT) in cancer patients undergoing bone marrow transplantation (BMT). However, donor T cell reactivity to host organs causes severe and potentially lethal inflammation, called graft-versus-host disease (GVHD). High dose steroids or other immune suppressives are used to treat GVHD that have limited ability to control the inflammation while incurring long-term toxicity. Novel strategies are needed to modulate GVHD, preserve GVT and improve the outcome of BMT. Regulatory T cells (Tregs) control alloantigen-sensitized inflammation of GVHD, sustain GVT and prevent mortality in bone marrow transplantation. Helminths colonizing the alimentary tract dramatically increase the Treg activity, thereby modulating intestinal or systemic inflammatory responses. These observations led us to hypothesize that helminths can regulate GVHD and maintain GVT in mice. Acute GVHD was induced in helminth (Heligmosomoides polygyrus)-infected or uninfected Balb/C recipients of C57BL/6 donor grafts. Helminth infection suppressed donor T cell inflammatory cytokine generation along with reduction in GVHD lethality and maintenance of GVT. H. polygyrus colonization promoted the survival of TGFβ generating recipient Tregs after a conditioning regimen with total body irradiation and led to a TGFβ-dependent in vivo expansion/maturation of donor Tregs after BMT. Helminths did not control GVHD, when T cells unresponsive to TGFβ-mediated immune regulation were used as donor T lymphocytes. These results suggest that helminths suppress acute GVHD, employing regulatory T cells and TGFβ-dependent pathways in mice. Helminthic regulation of GVHD and GVT through intestinal immune conditioning may improve the outcome of BMT.

Introduction

Graft versus host disease (GVHD) is a major and potentially severe complication of bone marrow transplantation. The disease is mediated by allo-reactivity of donor T lymphocytes to recipient major or minor histocompatibility antigens (1, 2). While the acute form of GVHD affects the skin, intestine and liver, chronic GVHD exhibits multi-organ infiltration similar to various autoimmune diseases (3).

Intestinal inflammation in GVHD simulates inflammatory bowel diseases (IBD), a group of immunological disorders that include ulcerative colitis and Crohn’s disease (CD). Furthermore, allelic variants of the mammalian receptor protein for bacterial muramyldipeptide, CARD15/NOD2, influences the propensity to develop CD as well as GVHD (4, 5). Certain genetic variants of IL23 receptor protect individuals from these two disorders (6, 7). Inflammation in mouse models of IBD or GVHD is controlled by various immune modulatory mechanisms that include regulatory T cells (Treg) that suppress inflammation driven by effector T lymphocytes (1, 4, 8, 9). Tregs express the transcription factor FoxP3 and contribute to intestinal immune regulation by cell contact-dependent mechanisms or by the production of modulating cytokines, such as IL10 and TGFβ (9–11). Helminthic regulation of intestinal immunity is associated with activation of Treg subsets, induction of regulatory cytokine production and depends on intact TGFβ circuitries (12–15).

The immune modulatory murine nematode Heligmosomoides polygyrus briefly resides in the sub-mucosa of the mouse duodenum after oral administration and then remains in intestinal lumen without causing systemic infection, until the adult worm is expelled. We have previously demonstrated that helminths like H. polygyrus trigger intestinal Treg activity and regulatory cytokine generation with consequent regulation of colitis in mice (13, 16). Other parasitic infestations may also have immune suppressive properties and have been shown to reduce clinical activity in conditions, such as multiple sclerosis, celiac disease and IBD (17–22).

Although GVHD can be prevented by depleting donor T cells from the graft, recipients then are predisposed to severe infectious diseases. Engraftment as well as the graft versus tumor (GVT) effect may be diminished by donor T cell removal (1, 23, 24). In preclinical mouse models, several laboratories have shown that GVHD can be prevented with preserved anti-tumor immunity (graft versus tumor; GVT) by co-administration of donor conventional T cells and Tregs given in equal numbers (23, 25–27). However, the production of high numbers of human Treg suitable for infusion remains a technically challenging goal.

In this study, we show that H. polygyrus treatment of the recipients protects mice from fatal acute GVHD and sustains GVT. Regulation of GVHD is associated with induction of Tregs that may regulate Th1 inflammation by means of TGFβ expression and secretion. Helminths reduce GVHD-related Th1 inflammation. Furthermore, in a TGFβ-dependent manner, H. polygyrus administration decreases GVHD-related mortality. Since the intestine is a primal organ for GVHD generation (5, 28), our results open the possibility that intestinal immune conditioning of patients prior to bone marrow transplantation (BMT) may be a useful strategy to reduce GVHD-related morbidity, mortality and still preserve donor graft’s antitumor immunity.

Materials and Methods

Mice, H. polygyrus administration and egg counting

We utilized wild type (WT) C57BL/6 (H2b) and Balb/C (H2d) mice (The Jackson Laboratory, Bar Harbor, ME) as well as a C57BL/6 mouse strain with a T cell-specific defect in TGFβ signaling (Cd4-TGFBR2 (Jackson Laboratories #005551; also named TGFβ receptor II dominant negative (TGFβ RII DN)) (H2b)(29). Five to six week old Balb/C mice were inoculated with 150 H. polygyrus third stage larvae (L3) by oral gavage. Infective H. polygyrus L3 (original specimens archived at the U.S. National Helminthological Collection no. 81930; also named H. polygyrus (bakeri) or H. bakeri in some publications (30, 31) were obtained from mouse fecal cultures of eggs by the modified Baermann method and stored at 4°C until used. The number of eggs in hydrated stool pellets was enumerated in duplicate at the indicated time points for each mouse and displayed as egg number/stool weight. Mice were housed and handled following national guidelines and as approved by our Animal Review Committee.

Cell purification for GVHD induction

Donor bone marrow (BM) cells were obtained from the femurs and tibias of uninfected, 5–8 week old C57BL/6 mice and T cells were depleted (TCD) using mouse panT cell beads (Dynal Biotech) according the manufacturer’s instructions. Donor T lymphocytes (CD3+) were magnetically enriched from splenic single cell suspensions of uninfected, 5–8 week old C57BL/6 and TGFβRII DN mice using the T cell untouched isolation kit (Miltenyi Biotech).

Cell purification for in vitro cultures

To determine TGFβ cytokine generation of Treg-enriched vs Treg-depleted cultures, CD4+ T cells were purified from splenic and mesenteric lymph node (MLN) single cell suspensions of H. polygyrus-infected and uninfected Balb/C mice using CD4 T cell untouched isolation kit (Miltenyi Biotech) and separated into CD25 positive and CD25 negative fractions using anti-CD25 PE labeling, followed by magnetic separation with anti-PE beads (Miltenyi Biotech). Enrichment or depletion efficiency was >98% with these techniques (data not shown). To determine helminthic regulation of donor T cell IFNγ TNFα and interleukin 4 (IL4) cytokine output during GVHD, donor CD3+ T cells were sorted from total anti-CD3 FITC and anti-H2b PE stained splenocytes of uninfected and H. polygyrus-infected Balb/C recipients 6 days after GVHD induction using FACS Vantage SE DiVa cell sorter (Becton Dickinson).

Total body irradiation (TBI) and GVHD induction

Our studies utilized an MHC I/II mismatch, acute lethal GVHD model(26). Three week helminth-infected and uninfected Balb/C recipients (H2d) underwent lethal total body irradiation from a Cs137 source (8.5 Gy in two divided doses given four hours apart) and were administered 10×106 T cell depleted bone marrow (TCD-BM) cells and 1.5×106 splenic T lymphocytes from uninfected C57BL/6 WT donors. To determine the effect of helminth infection on the conditioning regimen (TBI) without bone marrow transplantation, mice underwent irradiation with total doses ranging from sublethal 4 Gy to lethal 15 Gy according to the same protocol. Similar split irradiation doses were used, except that the 4 Gy group received a single dose. To characterize the role of TGFβ signaling in helminth-induced regulation of donor T cell-mediated GVHD, 1.5×106 donor splenic T cells from TGFβRII DN mice were administered along with 10×106 TCD BM cells from C57BL/6 WT mice into uninfected and 3 week H. polygyrus-infected Balb/C recipients. Mice were monitored daily for survival for up to 112 days in different experiments. The disease severity was scored daily based on animal weight, posture, activity, fur texture and skin integrity(32–34). In parallel experiments, uninfected and helminth-infected mice were sacrificed 6 days after GVHD induction for cellular and histological analysis.

Quantification of tumor load and assessment for GVT by bioluminescent imaging (BLI)

Luciferase expressing A20 leukemia/lymphoma cells (A20-luc) syngeneic with recipients (H2d) were used for these experiments (35). Each recipient mouse received 3×105 A20-luc tumor cells intravenously within 24 hours after BMT. Tumor load was assessed regularly in BMT recipient mice using an Ami 1000 Advanced Molecular Imager (Spectral Instruments, Tucson, AZ) live animal imaging system. Five minutes before BLI, mice were placed in an oxygenated isoflurane chamber and administered D-luciferin (Promega, Madison, WI) intraperitoneally. BMT recipient animals were imaged for 5 minutes and tumor load was quantitated by means of Living Image software v2.50 (Caliper Life Sciences).

Flow cytometry

Six days after GVHD induction, uninfected and H. polygyrus-infected mice were sacrificed. Spleen and MLN were isolated for cellular analysis. For surface staining, cells were suspended at 2×107 cells/ml in PBS with 2% FCS and Fc receptors were blocked with 2.4G2 mAb. Cells were stained with various combinations of anti-CD3 FITC, anti-CD3 PE-Cy7, anti-CD4 FITC, anti-CD4 PE-Cy7 (eBioscience), anti-latent TGFβ (LAP) PE (Biolegend), anti-H2b PE, anti-H2d PE, and anti-H2b APC (BD Biosciences). For the intracellular FoxP3 staining, cells were stained with anti-FoxP3 PE, FoxP3 PE-Cy7 or FoxP3 APC using FoxP3 staining buffer (eBioscience) according the manufacturer’s instructions.

In vitro cell culture and cytokine ELISA

To quantify TGFβ secretion of FoxP3+ Treg-enriched and Treg-depleted CD4 T lymphocytes, magnetically purified CD4+CD25+ or CD4+CD25- splenic and MLN cells from uninfected as well as H. polygyrus-infected Balb/C mice without GVHD were stimulated with plate-bound anti-CD3 and soluble anti-CD28 (each 1 μg/ml) (eBioscience) for 48 hours in cell culture medium with 1% FCS and 1 mg/ml BSA(13, 36). TGFβ cytokine concentration in acidified and re-alkalinized supernatants was determined using antibody pairs from R&D Systems according to manufacturer’s instructions. Results were displayed after subtracting the TGFβ concentration of culture supernatants from TGFβ concentrations of the culture media. To determine helminthic regulation of donor T cell IFNγ and TNFα secretion, sorted donor splenic T cells (CD3+ and H2b+) from uninfected and H. polygyrus-infected Balb/C mice with GVHD were stimulated with plate-bound anti-CD3 and soluble anti-CD28 (each 1 μg/ml) for 48 hours in lymphocyte growth medium containing 10% FCS(15). Supernatants were analyzed for IFNγ, TNFα and IL4 content using antibody pairs from R&D Systems. Similarly, sera isolated from uninfected and H. polygyrus-infected Balb/C mice 6 days after GVHD induction were analyzed for IFNγ and TNFα cytokines.

Histopathology

Six days after GVHD induction, colons, lungs and livers from uninfected or H. polygyrus-infected mice were fixed in 4% neutral buffered formalin, processed and 6 μm sections were stained with hematoxylin and eosin. Tissues were analyzed for GVHD-related inflammation and the severity of inflammation was scored in blinded fashion by ANH (32, 37–40). GVHD-related colitis was graded based on the degree of inflammation and the frequency of crypt apoptosis. Inflammation is graded as none (score: 0), mild (1), moderate (2), severe without ulcer (3) and severe with ulcer (4). Crypt apoptosis was graded as rare (score: 0), occasional per 10 crypts (1), few per 10 crypts (2), majority of crypts containing apoptotic bodies (3), majority of crypts containing more than one apoptotic bodies (4). The minimal score in this grading system for colonic disease was 0 and the maximum score 8. GVHD-related lung inflammation was graded based on the presence of perivascular cuffing, vasculitis, peribronchiolar cuffing and alveolar hemorrhage. The minimal score in this grading system for lung inflammation was 0 and maximum 4.

Statistical analysis

Differences in survival between groups were determined by Kaplan Meier’s log rank test. Differences in cell number and composition, serum cytokine content, differences in splenic donor T cell cytokine generation, differences in TGFβ cytokine output of in vitro stimulated cell cultures and histopathological GVHD scores between two groups were determined using Student’s t test. Differences in cell number and composition between multiple groups were analyzed by ANOVA.

Results

Helminth treatment of the recipient reduces GVHD

Acute GVHD was initiated in uninfected or 3 week Heligmosomoides polygyrus-infected irradiated Balb/C recipients by transfer of total splenic T cells and TCD-BM cells from uninfected donor C57BL/6 mice. Mice started to display signs of GVHD 4–5 days later with this regimen and GVHD was characterized by loss of activity, skin discoloration, hunched body posture, and bloody diarrhea(32). Beginning at this time, uninfected mice displayed severe GVHD compared to a relatively normal appearance of H. polygyrus-infected mice (Figure 1A). H. polygyrus colonization of the recipient was associated with a significant increase in survival (p <0.001) (Figure 1B). Although weight loss associated with GVHD was not different between uninfected and H. polygyrus-exposed recipients (Figure 1C), helminth-infected mice exhibited significantly decreased disease activity (p<0.005) (Figure 1D). Weight loss or significant disease activity were not seen in helminth-infected or uninfected Balb/C recipients that were administered T cell depleted bone marrow (TCD BM) without splenic T cells from uninfected C57BL/6 donors (Suppl. Figure 1A). To determine whether irradiation alters the parasite fecundity, 8–9 week old male WT Balb/C mice underwent lethal TBI (8.5 Gy) without BMT, 3 weeks after helminth infection. Stool egg counts were performed in no irradiation control and lethally irradiated mice prior to and 6 days after TBI. Stool egg counts were similar between irradiated mice and control animals that did not receive irradiation (Suppl. Figure 2).

Figure 1. Helminths regulate acute GVHD in mice.

Figure 1

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(A) Heligmosomoides polygyrus infection protects mice from the severe inflammation during acute GVHD. Six days after BMT, H. polygyrus-infected animals (H. polygyrus) displayed less skin discoloration or hunching body posture compared to uninfected mice (Uninfected) with representative examples displayed. (B) Kaplan Meier survival curves of H. polygyrus-infected (open squares) or uninfected (closed squares) Balb/C recipients transferred TCD-BM and total splenic T cells from C57BL/6 mice. Cumulative data from three independent experiments with 9 animals in uninfected and 10 mice in the H. polygyrus-infected group (p<0.0001). (C) The weight change in these mice during the entire follow-up of the survival experiment (p= not significant (NS)). (D) The disease score between the uninfected (N=9) and H polygyrus-infected mice (N=10) over the entire course of the survival experiment (p<0.005).

We sacrificed parallel groups of uninfected and H. polygyrus-administered mice at day six after GVHD induction and analyzed tissues by histopathology. Gut colonization with H. polygyrus was associated with reduced inflammatory infiltrates in the colon (mean inflammatory score, day 6 post BMT, 2.4±0.8 in H. polygyrus-infected mice vs. 5.6±1.3 in uninfected mice; N=10/group for uninfected and helminth-infected; p<0.001) (Figure 2). No inflammation was evident in the large intestine of helminth-infected or uninfected Balb/C mice without bone marrow transplantation (BMT) (data not shown) or that underwent TCD BM (Suppl. Figure 1B). Numerous apoptotic bodies were evident in colonic samples from uninfected animals and not in samples from H. polygyrus-infected mice. Lung tissues from uninfected mice were characterized by dense mononuclear cell infiltrates as well as alveolar hemorrhages, while samples from H. polygyrus-administered animals showed fewer infiltrates with preservation of the air sacs (mean inflammatory score 1.3±0.5 in H. polygyrus-infected mice vs. 3.5±0.5 in uninfected mice; N=6/group for uninfected and helminth-infected; p<0.001) (Figure 2). No inflammatory changes were evident in the lungs of helminth-infected or uninfected Balb/C mice without bone marrow transplantation (BMT) (data not shown) or that underwent TCD BM (Suppl. Figure 1B). Liver tissues from uninfected or H. polygyrus-infected mice showed mild focal portal infiltrates with no difference between groups (data not shown).

Figure 2. Helminth infection is associated with decreased GVHD-related inflammatory changes in the colon and the lungs.

Figure 2

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Six days after BMT, colons from uninfected (A and B) and helminth-infected (D and E) mice were isolated, fixed and histopathological analysis was performed in 6 μm thick sections under low (10x) (A and D) and high (40x) (B and E) power of magnification. The amount of inflammation and the number of apoptotic bodies in intestinal epithelium (*) was significantly decreased in H. polygyrus–infected mice (D and E) compared to tissues from uninfected animals (A and B). (G) Severity of colonic inflammation in colons from uninfected and H. polygyrus-infected mice is graded as described in Materials and Methods. Representative images (A, B, D, E) and colitis scores (G) from 10 uninfected and 10 H. polygyrus-infected mice (dot plots, each dot representing colonic GVHD score from one mouse) from 3 independent experiments with the mean score (bar) (p<0.0001 btw. uninfected and helminth-infected). Similarly, lungs from uninfected (C) and helminth-infected (F) mice were isolated where histopathological analysis was performed in 6 μm thick sections under low power magnification (20x). Severity of inflammation in the lungs is graded as described in Materials and Methods. Dense inflammatory infiltrates (*) in samples from uninfected animals (C) was significantly decreased in lungs from helminth-infected mice (F). Representative images (C and F) and inflammatory scores (H) of GVHD-related inflammation in the lung from 6 uninfected and 6 H. polygyrus-infected mice (dot plots, each dot representing lung GVHD score from one mouse) from 2 independent experiments and the mean(p<0.0001 btw. uninfected and helminth-infected).

Helminth infection is associated with the persistence of recipient T cells

At day six after GVHD induction, the spleen and MLN cells were analyzed for donor and recipient markers. Most splenic or MLN cells in uninfected or H. polygyrus-infected BMT mice were CD3 positive T lymphocytes (Figure 3). No B cells were seen by CD19 staining (Suppl. Figure 3). While >97% of splenic and 95% of MLN T cells were donor-derived in uninfected mice, 9±2% of splenic and 16±2% of MLN cells were H2d+ recipient cells in H. polygyrus-infected mice (Figure 3). These data suggested that helminths stimulated the survival of recipient T lymphocytes.

Figure 3. Helminths promote the survival of recipient T cells.

Figure 3

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(A) Splenic (upper row) and MLN (lower row) CD3+ T cells isolated 6 days after BMT from H. polygyrus-infected mice (right) display a population of cells that does not stain for the donor marker H2b, while T cells from uninfected recipients with GVHD (left) uniformly stain for H2b. The percentage of cells in the corresponding quadrants is shown. Representative example from multiple experiments. (B) Splenic (upper row) and MLN CD3+ T cells (lower row) isolated 6 days after BMT from H. polygyrus-infected mice (right) display a population of cells positive for the recipient marker H2d, while very few cells from uninfected recipients with GVHD (left) stain for the recipient marker. The percentage of cells in the corresponding quadrants is shown. Representative example from multiple experiments. (C) Statistical analysis of the percentage of splenic (left) and MLN (right) recipient T cells with N representing the number of mice utilized cumulatively in multiple experiments. Each dot represents the percentage of host cells in a mouse in the corresponding organ with the mean percentages displayed as bars (p<0.0001 for recipient splenic T cells and p<0.001 for recipient MLN T cells).

Recipient T cell survival during GVHD may be due to helminth-induced protection from the TBI or due to suppression of donor T cell attack. To distinguish between these possibilities, we measured splenic and MLN T cell number as well as composition after total body irradiation (TBI; conditioning regimen) without BMT. Eradication of T cells through TBI in uninfected mice was dose dependent, with <20,000 splenic and <1,000 MLN T cells surviving 8.5 Gy TBI, the dose utilized in BMT experiments (Figure 4). Similarly, <2000 splenic and <100 MLN FoxP3+ Tregs survived 8.5 Gy lethal TBI without BMT. By contrast, T cells from helminth-infected mice survived 8.5 Gy and higher doses (11 and 15 Gy) of TBI though the number of surviving T cells and Tregs gradually decreased with the increase in radiation dose (Figure 4). Thus, helminths promoted the survival of host T cells and host FoxP3+ CD4 Tregs to the conditioning regimen, making host Tregs constitute a dominant regulatory T cell pool in the early period after BMT (see also Table 3).

Figure 4. Helminths promote the survival of recipient T cells and FoxP3+ CD4 Tregs after TBI.

Figure 4

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Spleen and MLN cells were isolated from uninfected or 3 week H. polygyrus-colonized mice 6 days after TBI at the indicated doses. Cells were stained for CD3, CD4 and FoxP3. (A) Representative examples of splenocyte and MLN cell CD3 staining after TBI with different doses. (B) Representative examples of splenocyte and MLN cell CD4 and FoxP3 staining after TBI with different doses. Cells were gated on CD3+ lymphoid population. (C) The total number of splenic and MLN CD3+ T lymphocytes (upper graphs) or FoxP3+ CD4 Tregs (lower graphs) were calculated using the total number of cells isolated from uninfected (Uninfected) and H. polygyrus-infected (H. poly) mice 6 days after TBI, the percentage of CD3+ lymphocytes and the percentage of FoxP3 staining CD4 T cells. Spleens were analyzed individually. The MLN cell number per animal was calculated by dividing the cell number from pooled MLN by the number of mice in each sample. Each set of data is derived from at least 3 independent samples (experiments) for each group. Statistical analysis between groups was performed by ANOVA, as displayed in the figure.

Table 3.

Helminths increase the percentage and number of donor and recipient FoxP3+ CD4 Tregs.

Organ CD3+ T Cell Type FoxP3+ CD4 Treg Percentage FoxP3+ CD4 Treg Number
Uninfected H. polygyrus p value Uninfected H. polygyrus p value
Spleen (N=7*) Donor 0.62±0.25 1.52±0.83 p<0.05 1.5 ± 1.1 × 104 3.3 ± 1.7 × 104 p<0.05
Recipient 2.75±1.25 4.97±2.36 p<0.05 2.6 ± 2.2 × 103 18.0 ± 1.5 × 103 p<0.05
MLN (N=6*) Donor 0.4 ± 0.1 2.2 ± 0.9 p<0.001 0.3 ± 0.2 × 103 2.1 ± 1.0 × 103 p<0.01
Recipient 8.6 ± 3.8 14.1 ± 3.3 p<0.05 0.4 ± 0.3 × 103 4.7 ± 2.6 × 103 p<0.01
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*

N = The number of experiments utilizing a single spleen or cells from pooled MLNs from multiple mice. The percentages represent the percent of donor or recipient FoxP3+ CD4 Tregs among all donor or recipient CD3+ T cells. The number of FoxP3+ MLN donor or recipient Tregs per mouse was calculated using the total number of mice utilized in each experiment, the total number of cells isolated from pooled MLN cells and the percentage of FoxP3+ CD4+ cells gated on CD3+ lymphocytes.

Helminths regulate donor T cell cytokine generation but do not interfere with the engraftment or early in vivo expansion of donor CD3+ T cells

Regulation of GVHD may involve suppression of early donor T cell proliferation(37). The number of splenic or MLN donor T cells was not different in helminth-infected recipients compared to uninfected mice (Table 1). To determine the effect of helminth infection on donor T cell cytokine production, equal numbers of FACS-sorted splenic donor T cells from uninfected and H. polygyrus- infected recipients were isolated 6 days after GVHD induction and studied for in vitro cytokine output. Helminth infection stimulated donor T cell IL4 output and led to reduced anti-CD3/28-stimulated donor T cell inflammatory cytokines (IFNγ, TNFα) production (Table 2). A parallel decrease in serum IFNγ and TNFα was observed in helminth-infected mice (Table 2). Thus, H. polygyrus infection regulated donor T cells with suppression of inflammatory cytokine and stimulation of Th2 or regulatory cytokine production. Helminthic regulation of donor T cells did not suppress the engraftment and early expansion of donor T cells.

Table 1.

Donor T cell numbers in spleens and MLN of uninfected or H. polygyrus-colonized BMT recipients.

Uninfected H. polygyrus p value
Spleen (x106/mouse) 3.7±1.9 (N=10) 4.8±2.6 (N=10) NS
MLN (x105/mouse) 0.8±0.4 (N=4) 1.7±0.9 (N=5) NS
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Table 2.

Helminthic regulation of cytokine production during GVHD.

Uninfected H. polygyrus p value
Donor T cell IFNγ (ng/ml) 153.4±8.4 (N=3) 54.7±33.9 (N=3) p<0.05
Donor T cell TNFα (ng/ml) 8.0±0.3 (N=3) 5.1±0.6 (N=3) p<0.05
Donor T cell IL4 (ng/ml) 0.5±0.1 (N=3) 2.8±0.4 (N=3) p<0.05
Serum IFNγ (ng/ml) 2.0±0.12 (N=5) 0.37±0.07 (N=5) p<0.001
Serum TNFα (ng/ml) 1.9±0.3 (N=5) 0.7±0.1 (N=5) p<0.01
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Helminths increase the percentage and number of donor and recipient FoxP3+ Tregs

Helminths promote the survival of FoxP3+ regulatory T cells that have been shown to regulate GVHD(23). To determine whether helminthic regulation of GVHD is associated with the induction of regulatory T cells (Treg), we analyzed the percentage and total numbers of donor- or recipient-derived Tregs in the spleen and MLN of uninfected and H. polygyrus-infected mice 6 days after BMT. Helminth infection led to a significant increase in the percentage and number of donor as well as recipient FoxP3+ CD4 Tregs in the spleen and MLN (Table 3). These data suggested that helminth-induced protection from GVHD was associated with increased donor and recipient Tregs in lymphoid compartments. Induction of Tregs may be one of the mechanisms of helminthic regulation of acute GVHD, as donor CD25+ CD4+ T cells enriched for FoxP3+ Tregs regulated acute GVHD, when co-transferred with conventional T cells (Suppl Figure 4), confirming previous observations that studied regulation of GVHD by Tregs in adoptive transfer models (23).

CD4 T lymphocytes enriched for FoxP3+ Tregs generate more TGFβ than other peripheral CD4 T cells

Immune regulatory pathways involving TGFβ lead to peripheral induction and maintenance of Tregs (41) and may be essential in helminth-induced immune modulation(13, 14). We have shown before that H. polygyrus colonization stimulates T cell TGFβ generation that is essential for Treg functions, such as IL10 production (13). We studied whether Tregs that are increased during GVHD in helminth-infected mice generated more TGFβ on a per cell basis. Most FoxP3+ CD4 T cells are found in the CD25+ CD4+ T cell compartment. Plate-bound anti-CD3 and soluble anti-CD28-stimulated splenic and MLN CD4+CD25+ T cells from H. polygyrus-infected or uninfected Balb/C mice generated approximately two fold more TGFβ compared to the CD4+CD25− T cell fraction, as shown by ELISA from supernatants harvested 48 hours after stimulation (Figure 5). MLN T cell isolates from helminth-infected mice generated significantly more TGFβ compared to MLN T cell isolates from uninfected mice (Figure 5). TGFβ cytokine content in anti-CD3/28 stimulated T cell depleted parallel cultures was <20 pg/ml and did not increase with anti-CD3/28 stimulation (data not shown). These data suggested that during GVHD, helminths induced the proliferation, generation or promoted the survival of TGFβ producing FoxP3+ CD4 Tregs.

Figure 5. Helminths enhance TGFβ generation from Treg-enriched and Treg-depleted MLN CD4 T cells.

Figure 5

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TGFβ output of plate-bound anti-CD3 and soluble anti-CD28 stimulated splenic and MLN CD25-enriched or depleted CD4+ T cells from 3 week H. polygyrus-colonized (Hp) or uninfected (Uninf) Balb/C mice without BMT was measured by ELISA from the cell culture supernatants. Data are representative examples from at least 3 independent experiments (*p<0.05 between CD25 negative and CD25 positive CD4 T cells for each group; **p<0.01 and ***p<0.001 for corresponding MLN cell groups between uninfected and H. polygyrus-infected mice).

Helminth infection is associated with an increase in MLN donor and recipient Treg latent TGFβ (latency associated peptide; LAP) expression during GVHD

Prepro-TGFβ peptide is cleaved into N-terminal latency associated peptide (LAP) and C-terminal TGFβ protein after transcription and translation(42). LAP is expressed on T cells that may regulate immune responses in a TGFβ-dependent manner(43). Therefore, we studied latent-TGFβ (LAP) expression on donor and recipient Tregs during acute GVHD. LAP staining of FoxP3+ CD4 Tregs gave a separate and bright signal on flow cytometry with a significantly elevated mean fluorescent intensity (MFI) compared to Treg staining with an isotype antibody (data not shown). In splenic lymphocytes, LAP expression on donor and recipient FoxP3+ CD4 Tregs did not change significantly after helminth infection (Figure 6). However, in MLN cells, helminth infection increased LAP MFI on donor and recipient FoxP3+ CD4 Tregs (Figure 6). These results indicated that H. polygyrus infection promoted the induction and/or maintenance of FoxP3+ Tregs with enhanced TGFβ generation during acute GVHD.

Figure 6. Helminths induce FoxP3+ CD4 Treg LAP expression.

Figure 6

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Spleen and MLN cells from uninfected (Uninfected) and helminth-infected (H. polygyrus or H. poly) mice were stained for CD3, CD4, CD8, H2b, H2d and FoxP3 and LAP. Representative dot plots from spleen (A–B) and MLN (C–D) cells isolated from uninfected and H. polygyrus-infected mice 6 days after BMT. Cells are gated on donor (A and C) or recipient (B and D) CD3+ CD4 T cells where LAP and FoxP3 expression are displayed as dot plots with numbers representing the percentage of events in the corresponding quadrants. The graphs display the MFI of LAP staining on FoxP3+ CD4 Tregs with each dot representing the analysis from one spleen or pooled MLN cells (*p<0.05 btw uninfected and helminth-infected; N=number of experiments).

Helminths regulate GVHD in a TGFβ-dependent manner

TGFβ is a strong regulator of Th1 immunity (41, 44). As helminth infection suppressed Th1 inflammation during GVHD, thereby increasing T cells that secrete TGFβ and express LAP, we studied whether helminths regulated donor T cell-mediated GVHD in a TGFβ-dependent manner. In these experiments we utilized donor T cells from uninfected TGFβRII DN mice (H2b), in which the T cells are unresponsive to TGFβ-mediated immune regulation due to T cell-specific over-expression of a truncated TGFβ receptor II (29). Because the engineered CD4 promoter driving the truncated TGFβRII expression lacked the CD8 silencer, both CD4 and CD8 T cells in these mice are unresponsive to TGFβ-mediated immune regulation. Uninfected and H. polygyrus-infected recipient male WT Balb/C mice were given TCD-BM cells from uninfected 5–7 week old male C57BL/6 WT donors and splenic T cells from male 5–7 week old TGFβRII DN mice (Figure 7A). Other groups included uninfected Balb/C recipients given TCD BM cells from uninfected C57BL/6 mice and uninfected or H. polygyrus-infected recipient Balb/C recipients given TCD-BM and splenic T cells from C57BL/6 WT donors. Transfer of TCD-BM cells was associated with survival of 90% of mice, while GVHD was uniformly lethal in uninfected Balb/C recipients that also received C57BL/6 WT or TGFβRII DN splenic T cells (Figure 7A). Strikingly, 40% of H. polygyrus-infected recipients that received T cells from WT C57BL/6 mice survived the GVHD, while H. polygyrus infection did not protect against GVHD lethality in recipients that received TGFβRII DN splenic T cells (Figure 7A). Some of the helminth-infected surviving mice observed >200 days after GVHD induction have maintained 60–80% of their original weight and displayed mild clinical disease activity (data not shown). Together, these data suggested that helminth infection regulated GVHD in recipient mice in a TGFβ-dependent manner.

Figure 7. Helminths regulate donor T cell-mediated GVHD in a TGFβ-dependent manner.

Figure 7

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(A) Kaplan Meier survival curves of uninfected Balb/C recipients transferred TCD-BM cells (BM) only from uninfected C57BL/6 mice (WT B6) (N=10) (triangles), uninfected Balb/C recipients transferred TCD-BM cells (BM) and total splenic T cells (T) from uninfected C57BL/6 mice (N=10) (closed squares), uninfected Balb/C recipients transferred TCD-BM cells from uninfected C57BL/6 WT (B6 WT) and splenic T cells from uninfected TGFβRII DN (DN B6) mice (N=15) (closed circles), H. polygyrus-infected Balb/C recipients transferred TCD-BM and splenic T cells from uninfected C57BL/6 mice (N=10) (open squares) and H. polygyrus-infected Balb/C recipients transferred TCD-BM cells from uninfected C57BL/6 mice (BM) and splenic T cells (T) from uninfected TGFβRII DN mice (N=12) (open circles). Cumulative data from three independent experiments with multiple mice from each group (p<0.0001). (B) Spleen and pooled MLN cells were isolated from helminth-infected Balb/C (Hp-infected) recipients of C57BL/6 (B6) or TGFβRII DN (DN) splenic T cell donors 6 days after BMT (All recipient mice also received C57BL/6 WT TCD BM cells). The percentages of total and donor CD3+ T cells and FoxP3+ CD4 T cells were determined by flow cytometry. Each dot represents one experiment from a single spleen or pooled MLN. The percentage of donor CD3+ T cells in all CD3+ T cells (upper panels) or the percentage of donor FoxP3+ CD4 Tregs in all CD3+ T cells (lower panels) from spleen (left) or MLN (right) isolates are displayed with each dot representing the percentage from a single sample (*p<0.05 btw B6 and DN, N=number of experiments, NS: difference not significant).

As helminths increased the percentage and number of GVHD regulating donor Tregs, we investigated the role of TGFβ in donor Treg repopulation after GVHD. Splenic T cells from uninfected C57BL/6 WT or TGFβRII DN mice have been previously shown to contain comparable frequencies of FoxP3+ Tregs (45), which we also found in our experiments (6.7±0.7% FoxP3+ in TGFβRII DN (N=3) vs 5.5±0.9 in C57BL/6 WT (N=3), p: not significant). Comparable donor T cell frequencies were observed in helminth-infected Balb/C BMT recipients that received splenic T cells from uninfected C57BL/6 or TGFβRII DN mice (Figure 7B). However, the percentage of donor FoxP3+ CD4 Tregs was significantly lower in helminth-infected BMT recipients of TGFβRII DN T cell donors compared to recipients of WT T cell donors (Figure 7B). These results suggested that TGFβ induced during helminth infection plays an additional critical role in donor Treg expansion during GVHD.

Helminthic regulation of GVHD is associated with preserved antitumor immunity (graft versus tumor; GVT)

To determine whether helminthic immune suppression preserves the donor T cell antitumor immunity (graft versus tumor, GVT), we administered luciferase expressing leukemia/lymphoma cell A20 (A20-luc) within 24 hours to BMT recipients. Live animal imaging demonstrated that uninfected or helminth-infected mice that received TCD BM without donor T cells developed tumors and died due to tumor burden within the first 24 days after BM and tumor transfer (Figure 8). There was no difference in survival between these groups suggesting that helminth infection did not have a beneficial or worsening effect on tumor development. No tumor development was observed in uninfected or helminth-infected mice that received donor T cells besides TCD BM and A20 (Figure 8). While uninfected mice died of severe GVHD, 5 of 9 helminth-infected mice survived without evidence of tumor throughout experimentation (Figure 8). These results suggested that helminths regulated GVHD and preserved GVT.

Figure 8. Helminths regulate GVHD and preserve GVT.

Figure 8

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(A) Live animal imaging of all surviving mouse population from a single experiment at indicated time points with the bioluminescence scale kept the same at each time point for all groups. (B) Kaplan Meier survival curves of uninfected Balb/C recipients transferred TCD-BM cells (BM) from uninfected C57BL/6 mice (WT B6) and A20-luc tumor cells, uninfected Balb/C recipients transferred TCD-BM cells (BM), total splenic T cells (T) from uninfected C57BL/6 mice and A20-luc tumor cells, helminth (H. polygyrus)-infected Balb/C recipients transferred TCD-BM cells (BM) from uninfected C57BL/6 mice (WT B6) and A20-luc tumor cells, helminth (H. polygyrus)-infected Balb/C recipients transferred TCD-BM cells (BM), total splenic T cells (T) from uninfected C57BL/6 mice and A20-luc tumor cells (N=cumulative number of mice, p<0.001 btw uninfected and helminth infected mice that received TCD BM, splenic T cells and A20-luc; p:NS btw uninfected and helminth infected mice that received TCD BM and A20-luc). (C) Percent mortality associated with tumor development. Proportions represent number of mice with tumor divided by the total number of mice that died during the experiment.

Discussion

Helminth infection of the gut has been shown to suppress inflammation in various autoimmune, allergic, and immunological disorders (46). The major finding of the present study was that helminth infection also regulated inflammatory responses in an acute, lethal GVHD animal model, promoting the survival of H. polygyrus-administered recipients. H. polygyrus treatment resulted in regulation of donor T cell Th1 cytokine generation, regulation of colitis and lung inflammation and enrichment for donor Tregs that generate high quantities of TGFβ (43) and that regulate GVHD (23). Another novel finding of the study was helminth-induced survival of recipient T cell subsets after TBI that led to persistence of recipient T cells and Tregs during GVHD. Helminths stimulated MLN recipient Treg TGFβ secretion, where MLN recipient Tregs continued with enhanced TGFβ expression during GVHD. Helminths promoted the survival from GVHD in a TGFβ-dependent manner and preserved GVT.

TGFβ suppresses the Th1 cytokine-driven inflammation models in mice, like IBD, experimental allergic encephalomyelitis or the donor T cell-mediated acute GVHD (1, 42, 44). Induction of host TGFβ pathways is critical in helminthic regulation of intestinal immunity as shown by others and our group (13, 14). Helminths may trigger these regulatory pathways by increasing TGFβ production from host cells. Nematodes also produce factors with TGFβ-like activity that stimulate mammalian TGFβ signaling and induce T cell FoxP3 gene expression (14). In this study, we broadened these observations on helminthic immune regulation to another disease model, GVHD further indicating the importance of gut immune system and luminal microenvironment in the initiation and regulation of mucosal as well as systemic alloreactivity (28, 47–49). When we used donor T lymphocytes, that were unresponsive to TGFβ-mediated immune regulation, helminthic protection from lethal GVHD was abrogated, So, intestinal H. polygyrus larvae may regulate donor T lymphocyte alloreactivity through promoting host as well as donor T cell TGFβ synthesis or by means of generating TGFβ-like factors. These observations attest to a mechanistic link between helminthic stimulation of TGFβ pathway and suppression of acute GVHD.

One source of TGFβ in helminth-infected BMT recipients is the recipient Treg population. Recipient Tregs that survive the conditioning regimen may contribute to immune regulation after experimental hematopoietic stem cell transplantation (50). However, they are too few in number after lethal TBI and major mismatch BMT to help suppress acute GVHD. Here we show that helminths stimulate recipient Treg survival after TBI and BMT and augment recipient MLN Treg TGFβ expression before and after bone marrow transfer. TGFβ generating recipient Tregs may be a critical regulatory cell population in helminth-infected BMT recipients, as B cells are absent during this acute GVHD, and as helminths do not promote TGFβ generation by innate immune cells (51, 52). Besides regulating donor T cell Th1 alloreactivity, TGFβ generating recipient T cells and Tregs from helminth-infected mice may also facilitate donor T cell engraftment (53).

Helminth-induced TGFβ may regulate donor T cell Th1 inflammatory cytokine generation by directly inhibiting donor T cell Th1 differentiation (42) or by stimulating T cell or Treg IL10 production. Helminths induce T cell IL10 generation in a TGFβ-dependent manner (13, 26). Helminths may also regulate GVHD-related Th1 responses through induced Th2 cytokine generation, as we demonstrate here.

Besides regulating Th1 inflammation, TGFβ is also important for the induction and maintenance of Tregs, implying that helminthic induction of TGFβ may regulate GVHD through induction of various immune regulatory pathways, such as stimulation of donor, recipient Tregs and directly regulating Th1 responses. Previous studies have shown contradictory results on the role of TGFβ in Treg expansion (45, 54–57). No role for TGFβ was suggested, if T cell subpopulations were analyzed 6–8 weeks after experimentation, whereas TGFβ appeared critical in studies that examined Treg expansion within a few days after T cell transfer or very early in life. Consistent with these early response experiments, we demonstrated that TGFβ is critical for rapid and robust donor Treg expansion and is crucial in regulating acute lethal GVHD.

TGFβ is produced as prepro-peptide where the N terminal cleaved portion of the protein, called latency-associated peptide (LAP) is secreted from the cell, non-covalently attached to the cleaved C terminal part of the original prepro-peptide(42). The C terminal protein is the TGFβ cytokine. Separation of the non-covalently attached N-terminal LAP leads to activation of TGFβ, permitting the binding of TGFβ to the TGFβ receptor complex and triggering signal transduction. In addition to the secreted TGFβ cytokine, non-covalently attached LAP and TGFβ proteins are also present in membrane-bound forms on regulatory cell subsets that dampen immune responses in cell contact- and TGFβ-dependent manner (43). We found that H. polygyrus infection was associated with an increase in MLN donor and recipient FoxP3+ Tregs expressing LAP, and that protection from acute GVHD requires donor T cell TGFβ signaling. This confirms the importance of TGFβ in regulating intestinal immunity (58) and the importance of the gut immune system in regulating GVHD.

GVHD has remained a challenge of clinical practice with rising numbers of hematopoietic stem cell transplantation to treat various hematological or non-hematological diseases(24). Various studies have shown the importance of Tregs in regulating effector donor T cells and suppressing acute or chronic GVHD (23, 27, 59–62), with ex vivo Tregs being a new area of clinical investigation in bone marrow transplantation. Purification and in vitro expansion of Tregs to a sufficient dose to manage GVHD is a challenge in clinical practice. Therefore, the role of strategies to expand the Tregs in vivo is also investigated clinically (63). Our results suggest that in vivo induction of Tregs by self-limited colonization of the gut with helminths and utilization of TGFβ pathway may suppress donor T cell Th1 immune reactivity, enable regulated donor T cell engraftment and deserve to be investigated as a novel and cost effective tool to control GVHD.

Helminths have been used with success in patients to treat inflammation (22). Helminths may regulate immunity directly or through modulation of gut flora, enriching the intestinal microbiome for beneficial or probiotic strains (64), where GVHD is associated with major shifts in the composition of intestinal flora in animal models or patients (47). With recent evidence showing that helminth products may regulate inflammatory responses similar to helminth infections (65), exposure to helminths or helminth products may become a novel and safe therapy of GVHD with preserved antitumor immunity (GVT), allowing broader use of bone marrow transplantation.

Supplementary Material

1

Acknowledgments

Funding Information: This study was supported by K08 DK082913 from the NIDDK, Lymphoma SPORE P50 CA097274 from the NCI, ACS-IRG-77-004-31 from the American Cancer Society administered through the Holden Comprehensive Cancer Center at the University of Iowa (MNI), R01 AI34495, R01 HL56067, AI34495, and HL11879 (BRB) and by VA Merit (DEE).

Footnotes

The authors do not report any conflict of interest.

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NIHMS644178-supplement-1.pdf (199KB, pdf)

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