ALBERT

Description: INTRODUCTION: The intestinal microbiota is essential for the fermentation of fibers into the short-chain fatty acids (SCFA) butyrate, acetate and propionate. SCFA can bind to G-protein-coupled receptors GPR41, GPR43 and GPR109a to activate downstream anti-inflammatory signaling pathways. In colitis or graft versus host disease (GVHD), GPR43 signaling has been reported as an important regulator of intestinal homeostasis by increasing the pool of regulatory T cells. In contrast to GPR43, which binds preferentially propionate and acetate, GPR109a is the major receptor for butyrate. We and others have demonstrated that butyrate can ameliorate gastrointestinal injury during GVHD through enterocyte protection. Therefore, we hypothesized that GPR109a plays an important role in the pathophysiology of intestinal GVHD, focusing specifically on alloreactive T cells. METHODS AND RESULTS: Using mouse models of GVHD, we examined the role of the butyrate/niacin receptor, GPR109a in allogeneic hematopoietic cell transplantation (allo-HCT). First, we studied whether a genetic knock-out (KO) of GPR109a in transplant recipient mice affected GVHD, but GPR109a-KO recipient mice did not exhibit increased mortality from GVHD compared to wild type (WT) mice. We next investigated the role of GPR109a in the donor compartment by transplanting either BM or T cells from WT or GPR109a-KO mice into major MHC mismatched BALB/c host mice. Mice transplanted with B6 BM, with T cells from a GPR109a-KO mouse into BALB/c hosts displayed a lower incidence of lethal GVHD (Fig. 1A). To determine whether the attenuation of GVHD is intrinsic to GPR109a-KO T cells, we established BM chimeras and performed a secondary transplant by transplanting B6 BM + (B6 à Ly5.1) or (GPR109a à Ly5.1) T cells into BALB/c hosts. We observed the same improvement in survival in mice that received GPR109a-KO T cells. This indicates an intrinsic role for GPR109a specifically in the donor hematopoietic compartment. Having identified a T-cell specific requirement for GPR109a we next examined expression of GPR109a on WT T cells in vitro at baseline and following stimulation with CD3/28 and found GPR109a significantly upregulated on both CD4+ and CD8+ T cells after 72 hours of stimulation (Fig 1B). At steady state in vivo, we observed the same numbers and percentages of splenic effector memory, central memory, and naïve CD4+ T cells as well as regulatory T cells in WT B6 mice and GPR109a-KO mice, suggesting normal T cell development in the knockout mice. In an in vitro mixed lymphocyte reaction (MLR), GPR109a-KO CD4+ T cells become activated, proliferate, polarize and secrete cytokine (specifically IFNg) to the same level as WT CD4+ T cells, suggesting normal functional capacity. However, after allo-HCT in mice we observed significantly fewer CD4+ and CD8+ T cells, and specifically fewer effector memory CD4+ T cells (Fig. C), in the small and large intestines of mice that received GPR109a-KO T cells at day 7 post transplant. In contrast, we found significantly more regulatory T cells in the intestines (Fig. 1D) and the spleen of GPR1091-KO T cell recipients, while numbers and percentages of polarized Th1 and Th17 T cells were similar between the two groups. We further 16S rRNA sequenced the gut microbiota of mice at day 7 after transplant and observed an increased relative abundance of bacteria from the genus Clostridium (Fig. 1D) along with an increased concentration of cecal butyrate as measured by GC-MS (Fig. 1E). In a preliminary graft versus tumor (GVT) experiment, we found that mice that received A20 tumor cells and GPR109a-KO T cells exhibited increased survival compared to mice that received A20 tumor cells and WT T cells. These preliminary findings suggest that GPR109a-KO T cells maintain their graft versus tumor response while causing less GVHD, and exclude a defective functional capacity. CONCLUSIONS: We report a novel role of the butyrate/niacin receptor GPR109a on donor T cells in allo-HCT as a genetic knock-out on T cells attenuates lethal GVHD. As these T cells are tested as functionally intact, we propose that the reduction in overall T cells of KO T cell recipients may underlie the attenuation in GVHD. Furthermore, such a reduction in allograft-induced gut injury is accompanied by maintenance of the gut commensal Clostridium and butyrate production, which is known to protect the intestinal epithelium and increases the regulatory T cell pool. Disclosures No relevant conflicts of interest to declare.

Description: Introduction. Clinical graft-versus-host disease (GVHD) phenotypes can selectively involve the upper or lower tract and extraintestinal tissues and organs. High gut microbiota diversity and the abundance of certain bacterial commensals within the Clostridiales order are associated with decreased risk of GVHD-related mortality after allogeneic stem cell transplantation (allo-HCT). The microbiota-derived short-chain fatty acid butyrate has been found beneficial in reducing murine GI aGVHD. Conversely, microbiota disruption is associated with expansions of potentially pathogenic bacteria, including facultative anaerobes, and can worsen allo-HCT outcomes. Since the vast majority of the intestinal microbiota biomass resides within the colon, we hypothesized that certain features of the colonic microbiota confer local protection to the lower GI tract. Methods. We evaluated 216 patients (median age 55 years) who underwent unmodified allo-HCT and had stool samples collected under our institutional fecal microbiome biobank between 01/2011 and 02/2017. Patients were classified in three groups according to the organ involved in aGVHD: Upper GI only (UGI, n = 56), lower GI with or without upper GI involvement (LGI, n =61 ), no GI tract involvement (non-GI, n = 29). A fourth group of 70 control patients with no GVHD whatsoever were selected on the basis of the study inclusion and exclusion criteria and having evaluable samples in our biobank in a temporal distribution that matched onset of GVHD. Microbial diversity was computed using Simpson's reciprocal index. Results. A total of 902 stool specimens were analyzed with an average of 4.2 samples/patient. Samples were grouped into pre-onset (day -20 to day 0) and post-onset (day 1 to day 20) aGVHD. Trends in intestinal microbiota features relative to the day of aGVHD onset showed distinct dynamics among the groups. Prior to aGVHD onset, all groups showed similar microbial diversity, and similar abundance of anaerobic commensals, including members of genus Blautia (Fig 1A-C). After aGVHD onset, microbial diversity and commensal anaerobe abundance in LGI cases were significantly lower than non-GI (p = 0.01 and 0.008, respectively) and UGI (p = 0.03 and 0.03, respectively) groups (Fig. 1A, 1B). The LGI group also had significantly lower abundance of the genus Blautia (p = 0.02, Fig 1C). Conversely, the LGI group had increased prevalence of facultative anaerobes (LGI vs. non-GI, p = 0.03, Fig. 1D). Prior to aGVHD onset, the LGI cases had lower abundance of predicted butyrate-producing bacteria (LGI vs. no-GVHD, p = 0.03), which was maintained in all three GVHD groups post-onset aGVHD (Fig 1E). Conclusions. Patients without GVHD, non-GI and UGI aGVHD phenotypes had higher microbial diversity, abundance of anaerobe commensals including genus Blautia, and predicted butyrate-producing bacteria after aGVHD onset than patients with lower GI tract involvement by aGVHD. In contrast, the LGI cases had features consistent with microbiota dysbiosis including prevalence of facultative anaerobes, and lower abundance of butyrate-producing bacteria before and after aGVHD. These findings are consistent with a model in which a diverse microbiota abundant in potential beneficial commensals exerts a local protective effect in the lower GI tract and have potential practical implications in future prophylactic and therapeutic interventions in aGVHD. Figure 1 Disclosures Gomes: Seres Therapeutics: Other: Part of Salary. Slingerland:Seres Therapeutics: Other: Salary supported by Seres funding. Pamer:Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Seres Therapeutics: Honoraria, Patents & Royalties; MedImmune: Honoraria; Novartis: Honoraria; Ferring Pharmaceuticals: Honoraria. Peled:Seres therapeutics: Consultancy, Honoraria. van den Brink:Acute Leukemia Forum (ALF): Consultancy, Honoraria; Merck & Co, Inc.: Consultancy, Honoraria; Magenta and DKMS Medical Council: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Other: Licensing; Seres Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria; Therakos: Consultancy, Honoraria; Flagship Ventures: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Evelo: Consultancy, Honoraria.

Description: Hematopoietic cell transplantation (HCT) is a critical treatment of patients with high-risk hematopoietic malignancies, hematological deficiencies, and other immune diseases. In allogeneic HCT (allo-HCT), donor-derived T cells recognize host tissues as foreign, causing graft-versus-host disease (GVHD) which is a main contributor to morbidity and mortality. The intestine is one of the organs most severely affected by GVHD and research has recently highlighted the importance of bacteria, particularly the gut microbiota, in HCT outcome and in GVHD development. Loss of intestinal bacterial diversity is common during the course of HCT and is associated with GVHD development and treatment with broad-spectrum antibiotics. Loss of intestinal diversity and outgrowth of opportunistic pathogens belonging to the phylum Proteobacteria and Enterococcus genus have also been linked to increased treatment-related mortality including GVHD, infections, and organ failure after allo-HCT. Experimental studies in allo-HCT animal models have shown some promising results for prebiotic and probiotic strategies as prophylaxis or treatment of GVHD. Continuous research will be important to define the relation of cause and effect for these associations between microbiota features and HCT outcomes. Importantly, studies focused on geographic and cultural differences in intestinal microbiota are necessary to define applicability of new strategies targeting the intestinal microbiota.