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Disruption of the intestinal bacterial microbiota is frequently observed in the context of allogeneic hematopoietic cell transplantation (HCT) and is particularly pronounced in patients who develop graft-versus-host disease (GVHD). Donor fecal microbiota transplantation (FMT) restores gut microbial diversity and reduces GVHD in HCT recipients. The composition of the intestinal fungal community in patients with GVHD, and whether fungal taxa are transferred during FMT are currently unknown. We performed a secondary analysis of our clinical trial of FMT in patients with steroid-refractory GVHD with a focus on the mycobiota. We characterized the fecal mycobiota of 17 patients and healthy FMT donors using internal transcribed spacer amplicon sequencing. The donor who provided the majority of FMT material in our study represents an n-of-one study of the intestinal flora over time. In this donor, mycobiota composition fluctuated over time while the bacterial microbiota remained stable over 16 months. Fungal DNA was detected more frequently in baseline stool samples from patients with steroid-refractory GVHD than in patients with steroid-dependent GVHD. We could detect fungal taxa in the majority of samples but did not see evidence of mycobiota transfer from donor to recipient. Our study demonstrates the feasibility of profiling the mycobiota alongside the more traditional bacterial microbiota, establishes the methodology, and provides a first insight into the mycobiota composition of patients with GVHD.

Microbiota injury occurs in many patients undergoing allogeneic hematopoietic cell transplantation, likely as a consequence of conditioning regimens involving chemo- and radiotherapy, the widespread use of both prophylactic and therapeutic antibiotics, and profound dietary changes during the peri-transplant period. Peri-transplant dysbiosis is characterized by a decrease in bacterial diversity, loss of commensal bacteria and single-taxon domination (e.g., with Enterococcal strains). Clinically, deviation of the post-transplant microbiota from a normal, high-diversity, healthy state has been associated with increased risk of bacteremia, development of graft-versus-host disease and decreases in overall survival. A number of recent clinical trials have attempted to target the microbiota in allogeneic hematopoietic cell transplantation patients via dietary interventions, selection of therapeutic antibiotics, administration of pre- or pro-biotics, or by performing fecal microbiota transplantation. These strategies have yielded promising results but the mechanisms by which these interventions influence transplant-related complications remain largely unknown. In this review we summarize the current approaches to targeting the microbiota, discuss potential underlying mechanisms and highlight the key outstanding areas that require further investigation in order to advance microbiota- targeting therapies.

Human hematopoiesis is critically dependent on the transcription factor GATA2. Patients with GATA2 deficiency typically present with myelodysplastic syndrome, reduced numbers of monocytes, NK cells and B cells, and/or opportunistic infections. Here, we present two families that harbor distinct GATA2 mutations with highly variable onset and course of disease. We discuss the use of allogeneic hematopoietic cell transplantation in these patients, especially as treatment for pulmonary alveolar proteinosis.

Disruption of the intestinal microbiota occurs frequently in allogeneic hematopoietic cell transplantation (allo-HCT) recipients and predisposes them to development of graft-versus-host disease (GvHD). In a prospective, single-center, single-arm study, we investigated the effect of donor fecal microbiota transplantation (FMT) on symptoms of steroid-refractory or steroid-dependent, acute or late-onset acute intestinal GvHD in 15 individuals who had undergone allo-HCT. Study participants received a fecal suspension from an unrelated healthy donor via nasoduodenal infusion. Donor FMT was well tolerated, and infection-related adverse events did not seem to be related to the FMT procedure. In 10 of 15 study participants, a complete clinical response was observed within 1 month after FMT, without additional interventions to alleviate GvHD symptoms. This response was accompanied by an increase in gut microbial a-diversity, a partial engraft-ment of donor bacterial species, and increased abundance of butyrate-producing bacteria, including Clostridiales and Blautia species. In 6 of the 10 responding donor FMT recipients, immunosuppressant drug therapy was successfully tapered. Durable remission of steroid-refractory or steroid-dependent GvHD after donor FMT was associated with improved survival at 24 weeks after donor FMT. This study highlights the potential of donor FMT as a treatment for steroid-refractory or steroid-dependent GvHD, but larger clinical trials are needed to confirm the safety and efficacy of this procedure.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is often associated with chemotherapy- and radiotherapy-induced host tissue damage, leading to graft-versus-host disease (GVHD). Innate lymphoid cells (ILC) have an essential role in tissue homeostasis and tissue repair via their production of interleukin (IL)-22, which acts on intestinal stem cells. The tissue healing capacities of ILC via IL-22 in the context of allo-HSCT and GVHD has previously been demonstrated in a mouse model for acute GVHD. We investigated potential other ways of ILC-mediated tissue protection against GVHD. Tissue injury leads to the release of danger-associated molecular patterns (DAMPs). DAMPs interact with purinergic receptors and ectoenzymes on immune cells and induce pleiotropic effects, including activation of proinflammatory antigen-presenting cells and immunosuppressive effects via the generation of adenosine. Here, we report a novel subset of human ILC3 that coexpress the ectoenzymes CD39 and CD73 (ecto(+) ILC3). Ecto(+) ILC3 express RORγt and were present in the oral-gastrointestinal tract and bone marrow. ILC3 ectoenzyme expression is modulated by the proinflammatory cytokine IL-1β. Extracellular adenosine triphosphate (eATP) stimulated ecto(+) ILC3 to produce IL-22 and adenosine. Activated ecto(+) ILC3 suppressed autologous T-cell proliferation in coculture experiments via the production of adenosine. In allo-HSCT recipients, intestinal GVHD was associated with reduced proportions of ecto(+) ILC3 and decreased levels of adenosine and its metabolite inosine. Taken together, ecto(+) ILC3 have immunosuppressive properties, but in patients with GVHD, ecto(+) ILC3 are depleted. A lack of ecto(+) ILC3 and subsequent reduced capacity to neutralize DAMPs may contribute to the development of GVHD.

Introduction Enteric dysbiosis has a negative influence on the outcome of allogeneic hematopoietic stem cell transplantation (HSCT) recipients. The presence of an imbalanced microbiome, marked by decreased gut flora diversity and intestinal pathobiont domination, is associated with an increased risk to develop graft-versus-host disease (GvHD). Objectives This prospective, single-arm pilot study tested if donor fecal microbiota transplantation (FMT) is safe and effective to treat intestinal GvHD. Methods Fifteen patients with steroid-refractory or steroid-dependent GvHD received a single FMT via nasoduodenal infusion from an unrelated, healthy donor. Results FMT was well tolerated and there were no serious adverse events observed that could be attributed to FMT. With 6-month follow-up nearly completed for all patients, 11 participants showed a complete response (CR), defined as resolution of all GvHD symptoms four weeks after FMT, without other interventions to alleviate symptoms. In six patients, the normalization of stool frequency and consistency was sustained throughout follow-up and immunosuppressants were tapered successfully. Five other responders initially showed improvement of GvHD after FMT but relapsed upon prednisolone taper (CR/sf; complete responders with secondary failure). A durable response to FMT was associated with a better prognosis (Figure 1). 16S ribosomal RNA sequencing of fecal samples from the first 13 patients revealed overall low alpha diversity in patients pre-FMT (Figure 2). One week after FMT, the fecal microbial composition of CR patients resembled that of the donor the most (Figure 3). Analysis of fecal samples from the other patients will be completed in the near future and will give more insights into the effects on microbial diversity and composition that are introduced by donor FMT. Conclusion The encouraging data from this pilot study show the potential of donor FMT as a safe therapy for steroid-refractory or steroid-dependent GvHD and promote further investigation in larger cohorts.