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).

• Fecal microbiota transplantation (FMT) (Biological): The FMT capsules are prepared from healthy donor feces screened according to international consensus guidelines. Oral administration of human-derived gut microbiota capsules, each containing no fewer than 2×10¹¹ organisms, given at a dose of 20 capsules per day for 3 consecutive days (total 60 capsules per course).No dose adjustments will be made during the study. The total treatment duration is 3 days. This is a single-arm, open-label, phase I/IIa proof-of-concept safety exploratory trial. Participants will be followed for 3 months post-FMT to assess safety, feasibility, and preliminary biological signals of immune recovery.

Primary outcomes

• Composite Endpoint of Safety and Feasibility of Selective FMT in Post-Allo-HSCT Patients [3 months post-FMT]: Safety is assessed by the incidence, severity (CTCAE v5.0), and attribution of treatment-emergent adverse events (TEAEs) within 3 months post-FMT, with particular monitoring for grade ≥3 AEs, serious adverse events (SAEs), and FMT-related exacerbation of GVHD or bacteremia. Feasibility is measured by the FMT completion rate, defined as the proportion of enrolled patients who successfully receive ≥90% of the planned total dose (≥54 of 60 oral capsules). The study will be considered to have demonstrated safety and feasibility if: (a) the completion rate is ≥80% (lower bound of 95% CI >60%); (b) the incidence of FMT-related grade ≥3 AEs is ≤15% (upper bound of 95% CI <30%); and (c) no FMT-related SAEs occur that are definitely or probably attributed to the intervention. Independent safety review will be performed after every 5 patients, with predefined stopping rules.

Secondary outcomes

• Change in Fecal Secretory Immunoglobulin A (sIgA) Concentration [Baseline to 3 months post-FMT]: Fecal sIgA concentration (µg/g feces) will be measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit specific for human secretory immunoglobulin A. Stool samples will be collected at baseline (pre-FMT) and at 3 months post-FMT. The endpoint is defined as the absolute and percentage change from baseline to 3 months post-FMT, with a signal of immune reconstitution defined as an increase of ≥50% from baseline or normalization to ≥50 µg/g feces. The assay will be performed according to the manufacturer's instructions, and results will be expressed as µg per gram of feces.
• Change in Serum Immunoglobulin Levels [Baseline to 3 months post-FMT]: Serum IgA, IgM, and IgG concentrations (g/L) will be measured using a commercially available immunoturbidimetric or nephelometric assay on an automated clinical chemistry analyzer. Blood samples will be collected at baseline (pre-FMT) and at 3 months post-FMT. The endpoint is defined as the absolute and percentage change from baseline to 3 months post-FMT. Assays will be performed in the institutional clinical laboratory according to standard operating procedures, and results will be expressed as grams per liter.
• Change in Fecal Short-Chain Fatty Acid Concentrations [Baseline to 2 weeks post-FMT]: Fecal propionate and butyrate concentrations (µmol/g feces) will be quantified using gas chromatography-mass spectrometry (GC-MS). Stool samples will be collected at baseline (pre-FMT) and at 2 weeks post-FMT. Briefly, fecal samples will be acidified, extracted with organic solvent, and derivatized prior to GC-MS analysis. The endpoint is defined as the absolute and percentage change from baseline to 2 weeks post-FMT. The assay will be performed according to established laboratory protocols, and results will be expressed as µmol per gram of feces.
• Change in Peripheral Blood B-Cell Subsets [Baseline to 3 months post-FMT]: Peripheral blood B-cell subsets will be characterized by multi-parameter flow cytometry. Whole blood samples will be collected in EDTA tubes at baseline (pre-FMT) and at 3 months post-FMT. The following B-cell subsets will be quantified as percentages of total CD19⁺ B cells: plasmablasts (CD19⁺CD27ʰⁱCD38ʰⁱ), IgA⁺ B cells (CD19⁺IgA⁺), and exhausted B cells (CD19⁺CD21ˡᵒʷCD11c⁺). A minimum of 200,000 events will be acquired per sample using a flow cytometer. Data will be analyzed using FlowJo software. The endpoint is defined as the absolute and percentage change in each subset frequency from baseline to 3 months post-FMT.
• Donor Microbiota Engraftment Rate [3 months post-FMT]: Donor microbiota engraftment will be assessed by 16S rRNA gene amplicon sequencing of stool samples collected at baseline (pre-FMT) and at 3 months post-FMT. Bacterial DNA will be extracted using a commercial stool DNA extraction kit. The V4 region of the 16S rRNA gene will be amplified using barcoded primers and sequenced on a next-generation sequencing platform (e.g., Illumina MiSeq) targeting a minimum depth of 20,000 reads per sample. Raw sequences will be processed using a standardized bioinformatics pipeline (e.g., QIIME2/DADA2) to identify amplicon sequence variants (ASVs). Donor-unique ASVs are defined as ASVs present in the donor sample but absent in the recipient's baseline sample (relative abundance threshold >0.1%). Engraftment rate will be quantified as the proportion of donor-unique ASVs detected in recipient fecal samples at 3 months post-FMT, calculated using the Jaccard similarity index. The endpoint is defined as the percentage of donor-unique ASVs successfully engra

Sex: All, ages: 18 Years to any

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

• Results publication: Shin JH, Tillotson G, MacKenzie TN, Warren CA, Wexler HM, Goldstein EJC. Bacteroides and related species: The keystone taxa of the human gut microbiota. Anaerobe. 2024 Feb;85:102819. doi: 10.1016/j.anaerobe.2024.102819. Epub 2024 Jan 10. PMID 38215933
• Results publication: Qu S, Gao Y, Ma J, Yan Q. Microbiota-derived short-chain fatty acids functions in the biology of B lymphocytes: From differentiation to antibody formation. Biomed Pharmacother. 2023 Oct 27;168:115773. doi: 10.1016/j.biopha.2023.115773. Online ahead of print. PMID 39491858
• Results publication: Goguyer-Deschaumes R, Waeckel L, Killian M, Rochereau N, Paul S. Metabolites and secretory immunoglobulins: messengers and effectors of the host-microbiota intestinal equilibrium. Trends Immunol. 2022 Jan;43(1):63-77. doi: 10.1016/j.it.2021.11.005. Epub 2021 Nov 27. PMID 34848167

• Study Protocol (PDF, 2026-08-07)