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

Yue Li; Hung-lin Chen; Nadine Bannick; Michael D. Henry; David E. Elliott; George J Weiner; Bruce R. Blazar; M. Nedim Ince

doi:10.1182/blood.V124.21.1102.1102

Abstract 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 3rd 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. Disclosures No relevant conflicts of interest to declare.

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

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