Safety and Feasibility of FMT for Immune Recovery After HSCT

BACKGROUND Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for various hematological malignancies. However, B-cell-mediated humoral immune reconstitution after allo-HSCT is remarkably slow and often incomplete. Immunoglobulin M (IgM) typically recovers within 2-6 months, immunoglobulin G (IgG) within 3-18 months, while immunoglobulin A (IgA) recovery may be delayed up to 3 years or longer. Functional humoral immune recovery often requires more than 5 years. Previous studies have shown that acute and chronic graft-versus-host disease (GVHD) are significant contributors to persistent IgA deficiency.

The conditioning regimen prior to transplantation causes profound disruption of the gut microbiota, impairing the bidirectional regulatory network between the microbiome and host immunity. Microbial metabolites, particularly short-chain fatty acids (SCFAs) such as propionate and butyrate, play essential roles in B-cell differentiation and antibody production. Propionate is critical for inducing and maintaining intestinal regulatory T cells (Tregs), while butyrate provides energy for B-cell differentiation and immunoglobulin secretion.

Preliminary clinical data from the investigators' group show that 100% (5/5) of immunodeficient patients post-allo-HSCT exhibit specific propionate deficiency, whereas patients with general dysbiosis primarily show butyrate deficiency. This suggests that the propionate synthesis pathway, driven predominantly by Bacteroidetes, is more severely destroyed under the extreme post-transplant environment.

Based on ecological keystone species theory, propionate-producing bacteria (particularly Bacteroides species) are highly connected nodes in the gut microbial network. A cross-feeding relationship exists between propionate- and butyrate-producing bacteria, where the former degrade complex carbohydrates to provide growth substrates for the latter. The investigators propose the "propionate first, butyrate follows" two-stage synergistic reconstruction hypothesis:

Stage 1 (Microenvironment Preparation): Propionate-producing bacteria are preferentially restored, increasing intestinal propionate levels, inducing Treg cells, and creating an immune-permissive environment.

Stage 2 (Effector Activation): Butyrate-producing bacteria successfully colonize in the improved microenvironment, restoring butyrate synthesis and driving B-cell differentiation and sIgA secretion.

A prior Phase II trial by Rashidi and colleagues (2023) demonstrated that oral fecal microbiota transplantation (FMT) is safe and feasible in allo-HSCT patients and may reduce GVHD risk. Based on this evidence and the novel hypothesis, the investigators propose an innovative selective FMT strategy using a "super donor" matching approach.

Study Design This is a two-phase, single-center study conducted at The First Affiliated Hospital of Soochow University.

Phase 1 (Prospective Observational Cohort):

Allo-HSCT patients are enrolled and followed pre-transplant, and at 1, 3, and 6 months post-transplant. At each visit, peripheral blood and fecal samples are collected for serum immunoglobulins, fecal sIgA, TBNK lymphocyte subsets, fecal 16S rRNA sequencing, quantitative SCFAs (propionate and butyrate), and dietary assessment. At 6 months post-transplant, patients are stratified into three groups based on serum IgA levels: immunodeficiency (IgA <0.07 g/L with IgG/IgM not recovered), delayed recovery (0.07 g/L ≤ IgA ≤ 0.8 g/L, or IgA <0.07 but IgG/IgM recovered), and normal recovery (IgA >0.8 g/L with IgG/IgM normal). Extreme phenotype analysis (immunodeficiency vs. normal recovery) will be used for microbiota and metabolite comparisons.

Phase 2 (Single-arm, Open-label, Phase I/IIa Proof-of-concept Safety Trial):

Patients from the immunodeficiency group at 6 months post-transplant who meet additional inclusion criteria are enrolled. A baseline comprehensive assessment is performed to map the microbiota-metabolite deficiency profile, followed by matching with a donor whose gut bacteria are rich in both propionate-producing and butyrate-producing strains. Participants receive oral FMT capsules (≥2×10¹¹ organisms/g), 20 capsules daily for 3 consecutive days (total 60 capsules per course). Follow-up visits occur at 2 weeks, 1 month, 2 months, and 3 months post-FMT, with immune parameters, microbiome profiling, SCFAs, and dietary assessment repeated at each visit.

Safety Monitoring Safety is monitored at each visit using CTCAE v5.0 criteria. Serious adverse events are reported to the Principal Investigator and Ethics Committee within 24 hours. Strict donor screening, exclusion of active GVHD, and emergency preparedness are implemented. If Grade 3 or higher FMT-related adverse events occur, treatment is suspended and a safety review is initiated.

Statistical Methods Phase 1 analyses include linear mixed-effects models for longitudinal trends, LEfSe for differential taxa screening, and random forest/LASSO regression for IgA prediction modeling. Phase 2 endpoints are analyzed using Wilcoxon signed-rank tests for paired comparisons and Spearman correlation for temporal associations. Missing data are handled using last observation carried forward. Two-sided tests with a significance level of 0.05 will be used. All analyses will be performed using R software (version 4.x or later).

Inclusion criteria

• Age ≥ 18 years
• Receiving first allogeneic hematopoietic stem cell transplantation (allo-HSCT) with myeloablative or reduced-intensity conditioning
• Able to complete scheduled follow-up visits and sample collections
• Willing and able to provide written informed consent

For Phase 2 (FMT intervention): participants must additionally meet all of the following criteria at 6 months post-transplant:

• Serum IgA < 0.07 g/L with IgG and IgM not yet recovered to normal range
• No active grade III-IV acute graft-versus-host disease (GVHD)
• Off systemic antibiotics for at least 2 weeks
• Willing to receive fecal microbiota transplantation (FMT) and sign dedicated informed consent for the intervention

Exclusion criteria

• Early post-transplant death or loss to follow-up
• Active grade III-IV acute GVHD (at time of screening)
• Continuous use of high-dose immunosuppressants (prednisone-equivalent dose > 1 mg/kg/day)
• Refusal to participate or inability to comply with study procedures

For Phase 2 (FMT intervention): participants meeting any of the following criteria will be excluded from the FMT phase:

• Gastrointestinal obstruction, perforation, or severe intestinal dysfunction
• Severe cardiac, hepatic, or renal insufficiency that would preclude safe FMT administration
• Known allergy or hypersensitivity to any component of the FMT preparation