Intestinal Helminth Colonization Regulates Lethal Graft Versus Host Disease and Preserves Graft Versus Tumor in Bone Marrow Transplanted Mice

Introduction.Graft versus host disease (GVHD) is a frequent and severe complication of allogeneic bone marrow transplantation (BMT). Novel and less toxic treatment regimens are needed to suppress GVHD and preserve graft versus tumor (GVT). Self-limited intestinal helminth colonization stimulates host regulatory T cell (Treg) subsets. The intestine is a prime organ in GVHD generation, with Tregs playing a pivotal role in controlling GVHD. Treg-mediated suppression of GVHD preserves GVT. We hypothesized that self-limited intestinal colonization with helminths protects BMT mice from lethal acute GVHD and preserves the GVT.

Methods. Three weeks after administering 3^rd stage adult Heligmosomoides polygyrus larvae into 5-6 week old male WT Balb/C recipient mice (H2d), we initiated H2b→H2d MHC I/II mismatch model of acute GVHD by total body irradiation (TBI; 850 cGy) and administration of total splenic T cells and T cell depleted (TCD) bone marrow (BM) cells from male C57BL/6 WT (MHC: H2b) donors into uninfected control and helminth-infected male wild-type (WT) Balb/C (MHC: H2d) bone marrow recipients. In certain experiments, we used donor T cells from TGFβ RII dominant negative (DN) (TGFβ RII DN) mice (MHC: H2b) whose T cells are unresponsive to TGFβ-mediated immune regulation due to over-expression of a truncated TGFβ receptor II. Th1 (IFNγ, TNFα) cytokine generation was analyzed by ELISA. Splenic and mesenteric lymph node (MLN) cells were stained for H2b, H2d, T cell surface markers and FoxP3. Tissues were analyzed for GVHD-related inflammation. GVT was assessed in uninfected and helminth-infected BMT recipients by administration of luciferase expressing A20 leukemia/lymphoma cells (A20-luc; H2d) and IVIS imaging. Statistical and survival difference between groups was determined by Student’s t-test and Kaplan Meier curves, respectively.

Results. Helminths increased ~3-fold CD4 T cell and FoxP3+ Treg TGFβ generation (p<0.01 btw uninfected and helminth-infected). Helminths suppressed donor T cell Th1 cytokine production (p<0.05 btw control and helminth-infected mice (N=3)) and regulated clinical as well as histopathological (colon and lung) GVHD score (p<0.05 between control and helminth-infected, for each parameter from multiple experiments). Helminths promoted the survival of recipient T cells after TBI (Spleen control: 1.4±0.9 x 10⁴ vs. helminth-infected: 11.0±6.9 x 10⁴ CD3+ T cells (N=8; p<0.05); MLN control: 0.09±0.09 x 10⁴ vs. helminth-infected: 6.0±5.2 x 10⁴ (N=8; p<0.001)). Surviving T cells in H. polygyrus-colonized mice were enriched for FoxP3+ Tregs (spleen: 1.16±0.6 x 10⁴ and MLN: 0.65 ±0.56 x 10⁴ FoxP3+ Tregs). Recipient T cells enriched for Tregs survived during GVHD, as analyzed 6 days after BMT (3.3±1.1% of splenic and 15.9±3.5% of MLN host T cells being FoxP3+ Tregs). The number of recipient T cells in uninfected BMT mice was low for further Treg analysis. Helminths increased the number of GVHD-regulating donor-derived H2b+ FoxP3+ Tregs (Spleen: 66±32 x 10³ (control) vs. 206±73 x 10³ (helminth-infected) Tregs per mouse (p<0.05; N=6); MLN: 1.6 ±1.1 x 10³ (control) vs. 4.9±2.0 x 10³(helminth-infected) Tregs per mouse (p<0.05; N=4). Helminth infection was associated with >15 weeks survival of 40% of recipients, while all uninfected BMT recipient mice died of GVHD (p<0.01 between uninfected (N=10) and helminth-infected (N=10) WT recipients). Helminths did not promote the survival of recipients, when T cells unresponsive to TGFβ were used as donor lymphocytes. Helminths preserved the GVT in BMT mice transferred A20-luc. All uninfected and helminth-infected TCD BM transferred recipients died within few weeks displaying significant luciferase activity, while no luciferase signal was evident at day 70 in surviving (5/9) helminth-infected mice administered TCD BM and splenic T cells along with A20-luc. Uninfected mice (9/9) transferred splenic T cells, TCD BM and A20-luc died of GVHD without showing luciferase activity.

Conclusions. Our results show that (1) helminths stimulate TGFβ producing host T cells and FoxP3+ Tregs that survive BMT conditioning with TBI; (2) helminths regulate GVHD by inducing donor Tregs and in a TGFβ dependent manner; (3) helminths preserve GVT. With a safe side-effect profile, helminths or helminth products have the potential to be investigated as an alternative strategy to prevent or treat GVHD.