The Role of the Gut Microbiota in Allogeneic Hematopoietic Cell Transplantation

ABSTRACT

Allogeneic hematopoietic cell transplantation (allo‐HCT) is an effective treatment for patients with high‐risk hematologic malignancies. Over the last decade, gut microbiota composition during allo‐HCT has been associated with patients' outcomes. Treatment‐related factors, in particular the use of broad‐spectrum antibiotics and the conditioning regimen, frequently induce gut dysbiosis, which is associated with immune dysregulation, toxicity, and adverse outcomes, particularly after allo‐HCT. Microbial metabolites further modulate immune responses and therapeutic efficacy. Emerging microbiota‐targeted strategies—including antibiotic stewardship, nutritional interventions, probiotics, fecal microbiota transplantation, and postbiotics—show promise in reducing graft‐versus‐host disease, controlling inflammation, and improving treatment responses.

1. Introduction

Allogeneic hematopoietic cell transplantation (allo‐HCT) remains the standard of care and the only curative option for many hematologic malignancies. Over the past three decades, advances in HLA typing, donor selection, supportive care, and immunosuppressive strategies have markedly improved transplant outcomes and expanded eligibility to older patients and those with significant comorbidities. The therapeutic effect of allo‐HCT derives from two complementary mechanisms: cytoreduction achieved through a conditioning regimen and the immunologically mediated graft‐versus‐tumor (GVT) effect exerted by donor immune cells. Conditioning regimens vary in intensity along a continuum from myeloablative to reduced‐intensity and nonmyeloablative approaches. Myeloablative conditioning provides potent antitumor cytotoxicity but is associated with greater early toxicity and non‐relapse mortality, whereas reduced‐intensity strategies rely more heavily on the GVT effect and have enabled transplantation in older or medically frail patients. Selection of conditioning intensity reflects a complex balance between disease risk, relapse probability, patient age, comorbidities, and overall transplant fitness.

Despite its curative potential, allo‐HCT remains associated with significant short‐ and long‐term complications. Among these, graft‐versus‐host disease (GVHD) is the defining immunologic toxicity of transplantation and a major contributor to morbidity and mortality. Acute GVHD (aGVHD) classically involves the skin, gastrointestinal (GI) tract, and liver and results from early donor T‐cell–mediated inflammatory injury. Chronic GVHD (cGVHD) represents a distinct, often multisystem immune dysregulation syndrome with autoimmune and fibrotic features, and it is a leading cause of late non‐relapse mortality and impaired quality of life among survivors. Additional transplant‐related complications include infectious risks related to prolonged immune dysfunction, organ toxicities from conditioning regimens, graft failure, and late effects such as secondary malignancies and metabolic or cardiovascular complications. Finally, relapse of the underlying disease remains the leading cause of allo‐HCT failure, in particular in patients with high‐risk hematological malignancies.

To achieve safe and durable therapeutic outcomes, the immune system and the balance between GVT and GVHD are of utmost importance after allo‐HCT. The effective functioning of this immune response seems to be closely associated with the gut microbiota. Accumulating evidence points to a complex, bidirectional interplay among intestinal inflammation following cellular therapy, gut microbial composition, and immune homeostasis [1]. In particular, changes in the abundance of specific bacterial taxa, both before and after treatment, may serve as potential biomarkers or therapeutic targets. Overall, data support an association between gut microbiome dysbiosis and outcomes after allo‐HCT; nevertheless, it remains controversial whether the microbiome plays a causal role or merely reflects disease severity and clinical status. Consequently, additional mechanistic insights are expected to support the clinical relevance. This review will present the available evidence on the host–microbiome interactions in the context of allo‐HCT. In the first part, we will review the available data on microbiota in allo‐HCT, its role in intestinal barrier function, and its interplay with the immune system. Secondly, we will review potential strategies to modulate microbiota in those patients in order to improve their outcomes. Of note, this review manuscript focuses on adult patients, and evidence in pediatric patients is out of the scope of our review.

2. The Microbiota in Allo‐HCT

2.1. Allo‐HCT and Its Impact on the Microbiota

Allo‐HCT represents a cornerstone of cellular therapy and is extensively used in various hematologic malignancies [2]. However, the transplantation process is frequently accompanied by disruption of the GI mucosal barrier, primarily due to conditioning regimens, dietary alterations, and broad‐spectrum antibiotic use. These factors contribute to impaired colonization of the commensal gut microbiota, resulting in dysbiosis and reduced microbial diversity [2, 3]. Conditioning regimen intensity has been directly associated with gut microbiota diversity loss, with high‐intensity conditioning regimen being associated with the greatest injury, while nonmyeloablative regimen had a low‐grade injury [4]. Emerging evidence indicates that such disturbances in the gut microbiota are closely linked to a range of transplantation‐related complications, including GVHD, infections, and disease relapse, which are major contributors to early mortality following allo‐HCT [5].

2.2. Association Between Dysbiotic Microbiota and Complications After Allo‐HCT

In healthy individuals, the gut microbiota is characterized by high diversity and compositional stability. It is predominantly composed of Firmicutes and Bacteroidetes, which together account for approximately 90% of the total microbial population [6]. Following allo‐HCT, particularly during the early post‐transplant period, patients experience a pronounced loss of microbial diversity accompanied by significant alterations in microbial composition. This dysbiosis is marked by the overrepresentation of potentially pathogenic taxa, notably members of the class Bacilli, including Enterococcus, Streptococcus, and Lactobacillus spp.—as well as members of the class Gammaproteobacteria, such as Enterobacteriales and Pasteurellales [7, 8]. In contrast, beneficial obligate anaerobic commensals, particularly species of the genus Bacteroides and members of the order Clostridiales, are substantially depleted during this period [1, 9].

Reduced gut microbial diversity is significantly correlated with adverse outcomes following allo‐HCT, including decreased overall survival (OS) [2, 10, 11], increased risk of aGVHD and aGVHD‐related mortality [10, 11], a higher incidence of infectious complications [12], and increased non‐relapse mortality (NRM) [10, 11]. The abnormal expansion of specific pathogenic bacteria often signals poor clinical prognosis. Notably, the overgrowth of Enterococcus has been strongly associated with multiple negative outcomes, including reduced OS [13, 14], increased incidence of aGVHD [9, 13], higher aGVHD‐related mortality [15], and an elevated risk of bloodstream infections [14, 15]. Similarly, Staphylococcaceae have been closely linked to the development of GI aGVHD [1], and Streptococcaceae are associated with infectious complications [16].

A higher relative abundance of Clostridiales has been identified as a potential protective factor associated with improved OS [17]. Numerous studies have reported that these bacteria are significantly linked to a reduced risk of aGVHD [18, 19] and lower NRM [20]. Lactobacillus spp. have also been implicated in favorable outcomes [21, 22], likely due to their anti‐inflammatory effects and ability to promote mucosal repair [23, 24]. In addition, increased abundance of Actinomycetaceae and Fusobacterium spp. has also been reported as potential indicators of improved prognosis [20, 25].

It should be noted that the roles of certain bacterial taxa remain controversial. For example, research on Bacteroidales has produced inconsistent findings: some species from the genus Parabacteroides may exacerbate aGVHD [9], whereas others, like Bacteroides ovatus , appear to have protected against aGVHD [26, 27]. B. fragilis exhibits a dual role: its overgrowth following azithromycin treatment has been associated with an increased risk of malignancy relapse [28], whereas Sofi et al. [29] reported that it protects gut integrity and reduces aGVHD. Artacho et al. [9] suggested that the pro‐GVHD effects may be attributable to specific Bacteroides variants at the amplicon sequence variant level, rather than to the whole genus. Similarly, Akkermansia muciniphila also shows conflicting roles. These contradictory results may be due to the different species and sequencing methods applied in the different studies [11]. A summary of these findings is presented in Table 1. Beyond composition, a recent study revealed location‐specific changes in the microbiome and associated gut metabolites in murine aGVHD and identified phenyllactate as a novel regulator of GI GVHD, providing new insights into GI aGVHD pathogenesis [56].

TABLE 1.

Interactions between gut microbiota and clinical outcomes after allo‐HCT. [Color table can be viewed at wileyonlinelibrary.com]

Phylum	Class	Order	Family	Genus/species
Firmicutes	Clostridia	Clostridiales aGVHD↓ [18, 19]；non‐relapse mortality↓ [20]；OS↑ [17]	Lachnospiraceae prognosis↑ [2]；maintain GI integrity [30]; promote immune reconstitution [30]	Blautia aGVHD↓ [11], OS↑ [31], aGVHD‐related mortality↓ [31]; GI‐aGVHD↓ [32], bacteraemia↓ [32]
				Eubacterium relapse↓ [33]; progression↓ [5]; MRD negativity↑ [33]
			Clostridiaceae	Butyricicoccus aGVHD↓ [9, 34]
			Ruminococcaceae OS↑ [35]；aGVHD↓ [1, 35]; GI GVHD [1]	Faecalibacterium aGVHD↓ [9, 36]; aGVHD‐related mortality↓ [36]; MRD negativity↑ [33]
			Peptostreptococcaceae aGVHD↓ [37]
			Erysipelotrichaceae aGVHD↓ [37, 38]
	Bacilli	Lactobacillales	Enterococcaceae aGVHD↑ [1]; GI aGVHD ↑ [1]	Enterococcus aGVHD↑ [9, 13]; OS↓ [13]；related to GI integrity and immune reconstitution [15]；aGVHD‐related mortality↑ [13]; bacteremia↑ [14, 39]
			Lactobacillaceae aGvHD↑ [35]; mortality↑ [35]	Lactobacillus aGVHD↓ [21]
			Streptococcaceae infectious complications↑ [16]	Streptococcus
		Bacillales	Staphylococcaceae aGVHD↑ [1, 40]； GI, and steroid‐refractory aGVHD ↑ [1]	Staphylococcus CD4 T‐cell recovery↓ [33]
Bacteroidetes	Bacteroidia	Bacteroidales	Bacteroidaceae	Bacteroides GI‐aGVHD↓( Bacteroides ovatus [26, 27]; Bacteroides T6SS [7]) aGVHD‐related mortality↓( Bacteroides ovatus [26]); gut integrity↑ ( Bacteroides fragilis [29]; Bacteroides T6SS [7]); aGVHD↓( Bacteroides fragilis [29]); colonic aGVHD↑ (Bacteroides thetaiotaomicron [41])
			Prevotellaceae aGVHD↑ [42]	Prevotella aGVHD↑ [43]; aGVHD↑ [42]; CR↑ [28]
			Muribaculaceae aGVHD↓ [44]; aGVHD↑ [30]
			Tannerellaceae	Parabacteroides LGI‐aGVHD↓ [27]; aGVHD↑ [9, 30]; time‐to‐event*↓ [45]
Proteobacteria	Gammaproteobacteria pulmonary complications↑ [46]； predictive of mortality [46]；OS↓ [17]	Enterobacteriales	Enterobacteriaceae aGVHD↑ [30, 37]; Infection↑ [47]	Escherichia coli aGVHD↑ [48]; bloodstream↑ [48, 49, 50, 51]
		Pasteurellales GI‐aGVHD↑ [52]
Actinobacteria	Actinobacteria	Actinomycetales	Actinomycetaceae OS↑ [2]
Fusobacteria	Fusobacteriia	Fusobacteriales	Fusobacteriaceae	Fusobacterium GI‐aGVHD↓ [25]
Verrucomicrobia	Verrucomicrobiae	Verrucomicrobiales	Akkermansiaceae	Akkermansia muciniphila aGVHD↑ [53]; aGVHD↓ [11, 54]; steroid‐refractory aGVHD↑ [55]

Note: ↓ indicates a decrease in risk; ↑ indicates an increase in risk. Green: associated with better outcomes; Yellow: associated with worse outcomes; Gray: association is debatable. Time‐to‐event*: timing of seven outcomes [overall survival (OS), non‐relapse mortality (NRM), relapse, acute GVHD (grades II–IV and III–IV), chronic GVHD, and oral chronic GVHD].

Abbreviations: aGVHD, acute GVHD; GI, gastrointestinal; GVHD, graft‐versus‐host disease; MRD, measurable/minimal residual disease; NRM, non‐relapse mortality; OS, overall survival.

2.3. Microbiota‐Mediated Intestinal Barrier Function After Allo‐HCT

Prior to allo‐HCT, recipients typically undergo conditioning with chemotherapy, with or without radiotherapy, to eliminate residual malignant cells, suppress host immune responses, and facilitate donor hematopoietic cell engraftment. However, these conditioning treatments can cause substantial injury to the intestinal epithelium. In particular, they can damage Paneth cells and reduce the production of antimicrobial peptides such as α‐defensins, thereby disrupting the intestinal microbial ecosystem [41, 57]. Conditioning can also impair epithelial tight junctions, leading to increased intestinal permeability [58]. In addition, the widespread use of antibiotics during transplantation further perturbs the gut microbiota, promotes the expansion of mucus‐degrading bacteria [41], and disrupts the physiological hypoxic environment maintained by the intestinal epithelium [59], collectively weakening the intestinal barrier. These alterations facilitate the translocation of lipopolysaccharide (LPS) and other microbe‐associated molecular patterns (MAMPs) and pathogen‐associated molecular patterns (PAMPs) across the epithelial barrier into the lamina propria, thereby amplifying innate immune activation and exacerbating inflammation and aGVHD.

Meanwhile, the gut microbiota plays a critical role in maintaining intestinal homeostasis and barrier integrity. For example, dietary fiber or polysaccharides can be fermented by intestinal microbes to produce short‐chain fatty acids (SCFAs), which help stabilize the intestinal epithelial barrier [59, 60, 61, 62]. Primary bile acids can be converted by the gut microbiota into secondary bile acids that maintain mucosal barrier integrity and promote epithelial regeneration through receptor‐mediated signaling pathways [63, 64]. In addition, tryptophan can be metabolized by intestinal microbes into indole derivatives, some of which function as ligands for the aryl hydrocarbon receptor (AhR), thereby regulating mucosal immune responses and contributing to the maintenance of intestinal homeostasis [65, 66, 67, 68, 69]. See Figure 1 for details.

FIGURE 1.

The role of the microbiome and metabolites in intestinal barrier function during transplantation. Dietary fiber and polysaccharides are fermented by the gut microbiota to produce SCFAs. SCFAs promote epithelial oxygen consumption and stabilize hypoxia‐inducible factor (HIF), thereby enhancing intestinal barrier‐protective programs. In addition, SCFAs strengthen tight junction function and attenuate barrier disruption. They also contribute to mucosal barrier protection through modulation of T‐ and B‐cell responses and help maintain intestinal integrity through inhibition of histone deacetylases (HDACs). Primary bile acids are converted by the gut microbiota into secondary bile acids, which preserve intestinal mucosal barrier integrity and prevent bacterial translocation through activation of the farnesoid X receptor (FXR). Moreover, bile acids promote intestinal epithelial regeneration through activation of Takeda G protein‐coupled receptor 5 (TGR5) in intestinal stem cells. Tryptophan is metabolized by the gut microbiota into indole derivatives. On the one hand, these metabolites activate the aryl hydrocarbon receptor (AhR), thereby stimulating local lymphocytes to produce interleukin‐22 (IL‐22), which in turn promotes epithelial repair, supports mucus barrier defense, and induces antimicrobial peptide expression. On the other hand, indole derivatives also enhance tight junction function in intestinal epithelial cells. IEC, intestinal epithelial cell; ISC, intestinal stem cell; NOTCH1, Notch receptor 1; SRC/YAP, SRC proto‐oncogene, non‐receptor tyrosine kinase/Yes‐associated protein; ZO‐1, zonula occludens‐1. [Color figure can be viewed at wileyonlinelibrary.com]

2.4. Gut Microbiota and Immune System Crosstalk

2.4.1. Acute and Chronic GVHD Pathophysiology

The pathophysiology of acute graft‐versus‐host disease (aGVHD) can be divided into three stages: the initiation phase, the T‐cell activation phase, and the effector phase [70]. During the initiation phase, conditioning treatments such as chemotherapy and/or radiotherapy cause tissue injury in the host. This damage triggers the release of danger signals, including damage‐associated molecular patterns (DAMPs) and pathogen‐associated molecular patterns (PAMPs), as well as inflammatory mediators such as tumor necrosis factor (TNF), interferon‐γ (IFN‐γ), and LPS. These signals promote activation of host antigen‐presenting cells (APCs). Concurrently, reduced microbiota diversity disrupts epithelial integrity and immune homeostasis. In the T‐cell activation phase, activated host APCs stimulate donor‐derived alloreactive CD4⁺ and CD8⁺ T cells. Finally, during the effector phase, these activated T cells, together with pro‐inflammatory cytokines, induce injury to epithelial tissues in target organs—including the GI tract, skin, and liver—through processes such as apoptosis and necroptosis, ultimately producing the clinical manifestations of aGVHD. Regarding cGVHD, dysregulated B‐cell signaling and immunoglobulin production, persistent activation of selected T‐cell subsets, regulatory T‐cell deficiency, and tissue fibrosis are central mechanisms underlying its pathophysiology [71].

2.4.2. Role of Microbial Metabolites

The gut microbiota ferments dietary polysaccharides to produce SCFAs, including butyrate, propionate, and acetate. These metabolites play a vital role in maintaining intestinal immune homeostasis through multiple regulatory mechanisms. Immunologically, SCFAs can modulate both the frequency and functionality of regulatory T cells (Tregs) [72], promote T helper (Th)1 cell interleukin (IL)‐10 production [73, 74], and stimulate CD4+ T cells and innate lymphoid cells (ILCs) to produce IL‐22 [75], which help to reinforce the integrity of the intestinal epithelial barrier. In addition to immune modulation, SCFAs contribute to mucosal defense by activating B cells to produce secretory immunoglobulin A (sIgA) [76], by promoting the expression of intestinal mucin MUC2, enhancing the protection of epithelial cells [77, 78]. This establishes a dual “immune‐physical” barrier that protects the gut. Additionally, butyrate has been shown to exert a unique protective effect in GVHD. It may achieve this by expanding Treg populations through acetylation of the Foxp3 promoter [79], reducing MHC class II expression on intestinal epithelial cells [80], and regulating metabolite sensor G‐protein‐coupled receptor 43 (GPR43) [81]. These combined actions help to prevent excessive immune activation.

Secondary bile acids produced by gut bacteria help regulate intestinal bacterial overgrowth and maintain mucosal barrier integrity through farnesoid X receptor (FXR) signaling [63]. The FXR antagonist ursodeoxycholic acid can inhibit T cell proliferation and lower the risk of aGVHD‐related mortality in patients [82, 83]. Additionally, shifts in gut microbiota composition can influence bile acid metabolism. Han et al. [84] reported that Firmicute‐mediated bile acid metabolism may decrease the incidence of aGVHD in patients undergoing allo‐HCT.

AhR ligands are produced via microbial metabolism in the gut, with Lactobacillus, Clostridium, Bacteroides, and Faecalibacterium species capable of converting tryptophan into AhR ligands [85]. Clinical studies in patients undergoing allo‐HCT have demonstrated that reduced AhR activity is associated with a higher incidence of aGVHD and decreased OS [86, 87]. Accumulating evidence suggests that AhR modulates host–microbiota interactions by regulating IL‐22 and other signaling pathways [65, 66, 67, 68, 69], and can ameliorate aGVHD by promoting the expansion of peripherally induced Tregs [88].

Trimethylamine N‐oxide (TMAO) is a metaorganismal metabolite generated via gut microbial production of trimethylamine (TMA) from dietary substrates (e.g., choline/carnitine), followed by host hepatic oxidation to TMAO. Several gut bacteria (including Anaerococcus hydrogenalis , Clostridium asparagiforme , C. hathewayi , and C. sporogenes ) have been reported to contribute to TMA production [89]. Wu et al. [90] demonstrated that TMAO promotes M1 macrophage polarization via NLRP3 activation, enhances Th1/Th17 responses, and exacerbates aGVHD in murine models.

2.4.3. Role of Immune Cells in GVHD

2.4.3.1. Th Cells and Tregs

The maintenance of intestinal mucosal immune homeostasis relies on a finely tuned balance between pro‐inflammatory Th cells, including IFNγ‐producing Th1 cells and IL‐17A/IL‐22‐producing Th17 cells; and anti‐inflammatory Th cells, such as IL‐10‐secreting Th2 cells and Tregs. aGVHD has been primarily associated with Th1 responses, where IFNγ produced by Th1 cells promotes their own differentiation and can directly damage the intestinal mucosa [91]. In contrast, cGVHD involves the balance between Th1 and Th17 cell [92, 93]. Also, after allo‐HCT, an unbalanced Treg/Th17 ratio may also exacerbate inflammation and increase aGVHD and cGVHD risks [94, 95, 96]. Conversely, Th2 cells exert protective effects by effectively suppressing aGVHD progression [97].

Tregs, as naturally occurring suppressive CD4⁺ T cells, play a central role in maintaining immune tolerance and are modulated by the gut microbiota and its metabolites [98]. Adoptive Treg therapy can preserve beneficial GVT effects while controlling excessive immune responses, demonstrating significant potential for aGVHD prevention and treatment. On the other hand, the lack of Treg leads to uncontrolled Th1/Th17 expansion, eventually increasing the risk of cGVHD [99].

2.4.3.2. MAIT

Mucosal‐associated invariant T (MAIT) cells are a distinct subset of innate‐like T cells capable of recognizing riboflavin metabolites from diverse bacteria and fungi [100]. MAIT cells influence transplant‐related outcomes by modulating intestinal barrier integrity, reducing donor Th1 and Th17 cell frequencies, and producing mucosal‐protective cytokines such as IL‐17A, thereby shaping the composition of the gut microbiota [101, 102, 103]. Studies in both murine models and patients have suggested that MAIT cells may confer protection against aGVHD [101, 104]. Additionally, higher MAIT cell counts have been associated with increased cytomegalovirus reactivation rates and reduced incidence of late bloodstream infections [105].

2.4.3.3. γδ T Cell

γδ T cells, which lack alloreactivity and therefore do not trigger GVHD, exert potent GVT effects and play a critical role in allo‐HCT. Clinical studies indicate that higher γδ T cell levels post‐HCT correlate with longer OS, reduced infection and aGVHD rates [101, 106, 107, 108, 109], demonstrating targeted activity against cytomegalovirus‐infected cells, highlighting their antiviral potential [106, 107]. Thus, γδ T cells represent a critical target for modulating immunity, controlling infection, and improving post‐HCT outcomes.

2.4.3.4. Dendritic Cells

Dendritic cells (DCs), as pivotal APCs, play central roles in both innate and adaptive immunity. On one hand, CD11c‐dependent DCs promote IFN‐γ–producing Th1 differentiation, thereby driving the initiation of aGVHD [110, 111]. During aGVHD, impaired DC antigen presentation further induces Treg exhaustion, which accelerates cGVHD progression and establishes a pathogenic vicious cycle [112]. On the other hand, DCs also display protective properties. DC‐derived IL‐27 p28 modulates the thymic Treg/effector T‐cell balance and alleviates aGVHD severity [113]. Post‐transplant reconstitution of plasmacytoid DCs (pDCs) reduces aGVHD incidence, improves OS, and lowers infection risk [114]. The intestinal microbiota modulate DC secretion of IFN‐I and IL‐10, thereby mitigating aGVHD risk [115], and enhance the intestinal barrier integrity by regulating IL‐17 and IL‐22 production [110].

2.4.3.5. ILCs And B Cells

ILCs and B cells are critical for maintaining intestinal mucosal barrier integrity. Both pre‐transplant ILC presence and post‐transplant ILC reconstitution are associated with mucosal inflammation and GVHD risk [116, 117, 118, 119, 120]. Additionally, commensal bacteria, including Escherichia coli , Bifidobacterium spp., and segmented filamentous bacteria, support mucosal barrier function by regulating IgA⁺ B cells, whose levels closely correlate with GVHD severity and clinical prognosis [36, 121, 122]. Together, these findings suggest that the gut microbiota may indirectly influence transplant‐related outcomes by modulating ILC and B cell function.

The crosstalk between the gut microbiota and the immune system is summarized in Table 2.

TABLE 2.

Regulation of transplant‐related immune cells by the microbiota/metabolites.

Immune cell	Associated transplant complications	Modulation by microbiota/metabolites	Proposed clinical impact
Th1	Donor Th1 cell–derived IFN‐γ drives GVT effects [91]. IFN‐γ promots of Th1 differentiation→ damages the intestinal mucosal barrier, aGVHD↑ [91]	Itaconate suppresses Th1 cell differentiation [123]	aGVHD↓
		Microbiota‐derived SCFAs promote Th1 cell IL‐10 production [73, 74]	Maintain intestinal homeostasis
Th17	cGVHD↑ [92, 93]；aGVHD↑ [124]	Specific gut microbial taxa drive IL‐17–producing Th17 cells and cooperate with eosinophils.	MM progression↑*
Th2	aGVHD↓ [97]
Treg	Restoration of post‐transplant immune function [125] Prevention and treatment of aGVHD [126]. Pre‐ or peri‐transplant depletion of Tregs → subsequent immunotherapeutic strategies.↑ [127, 128] Lack of Treg → uncontrolled expansion of Th1/Th17 → cGVHD↑ [99]	SCFAs modulate Treg differentiation [72]	Colonic immune homeostasis↑
		Microbiota induce Tregs [98]	aGVHD↓
		AhR blockade induces intestinal Treg expansion [88]	aGVHD severity↓; survival↑
Th17/Treg	unbalanced Th17/Treg ratio→aGVHD or cGVHD [94, 95, 96]	Microbiota‐Driven Treg/Th17 Imbalance [37, 129]	aGVHD↑
MAIT	Influence intestinal barrier integrity [102, 103] Reduction of donor Th1 and Th17 cell frequencies [103] Modulation of gut microbiota composition [100] Protection against aGVHD [100, 101, 104] GRFS↑; OS↑; DFS↑ [101] Cytomegalovirus reactivation rates↑; Infections↓ [105]	Bacterial species encoding riboflavin biosynthesis pathways stimulate MAIT cell activation [130]. Increased gut microbial diversity is associated with higher MAIT cell abundance [131].	OS↑; aGVHD↓
γδ T cell	GVT effects↑ [132] Higher γδ T cell levels post‐HCT → OS↑ [106]; infection rates↓ [108] Cytomegalovirus reactivation rates↓ [133, 134]	Intestinal microbiota composition influences the abundance of Vδ2 γδ T cells [131].	aGVHD↓
DCs	CD11c‐dependent dendritic cell antigen presentation promotes IFN‐γ–producing Th1 and CD8+ T‐cell responsesa → GVHD↑ [111] Dendritic cell‐derived IL‐27 p28 regulates T cell program in pathogenicity and alleviates aGVHD [113]. Post‐transplant pDC reconstitution → aGVHD↓, OS↑, infection risk↓ [114]. During aGVHD, impaired DC antigen presentation → Treg exhaustion, promoting cGVHD↑ [112]	Gut microbiota modulate dendritic cell function, promoting Th17 and ILC3‐derived IL‐17 and IL‐22 production [110].	Intestinal barrier integrity↑
		Butyrate conditions monocyte‐derived DCs to polarize naïve CD4+ T cells toward IL‐10–producing type 1 regulatory T cells [115].	aGVHD↓*
Innate lymphoid cells	Both pre‐transplant ILC presence and post‐transplant ILC reconstitution are associated with mucosal inflammation and GVHD risk [117, 120]	AhR signaling and SCFAs promote IL‐22 production by innate lymphoid cells [66, 68, 75].	Intestinal mucosal defense↑
B cells	B‐cell homeostasis is associated with the development of cGVHD [135, 136].	Gut microbiota drive IgA production by B cells, while IgA in turn shapes microbial community homeostasis [137, 138].	Long‐term reduction of IgA + B cells → GVHD↑* IgA + B cell↑ → RRM↑*

Note: ↓ indicates a decrease in risk; ↑ indicates an increase in risk.

Abbreviations: DFS, disease‐free survival; GRFS, graft‐versus‐host disease–free, relapse‐free survival; GVT, graft‐versus‐tumor; MM, multiple myeloma; RRM, relapse‐related mortality.

^*Indirect evidence.

3. Microbiota‐Targeted Interventions

The above studies suggest that the gut microbiota may represent a viable target for modulating outcomes after allo‐HCT. Current microbiota‐targeted therapeutic strategies can act on microbial energy sources (e.g., diet, nutritional support, prebiotics), the microbial community itself (e.g., antibiotic stewardship, probiotics, fecal microbiota transfer [FMT]), or microbial‐derived metabolites (postbiotics), as illustrated in Figure 2.

FIGURE 2.

Strategies to modulate the gut microbiota and their impact on intestinal immune homeostasis and clinical outcomes. [Color figure can be viewed at wileyonlinelibrary.com]

3.1. Antibiotic Stewardship

In patients undergoing allo‐HCT, approximately 20%–60% develop systemic infections.

Prophylactic and broad‐spectrum antibiotics played a vital role in preventing and controlling bacterial infections in this setting. In particular, randomized studies support the use of fluoroquinolone antibacterial prophylaxis during the conditioning and pre‐engraftment neutropenic phase of allo‐HCT with consistent reductions in febrile neutropenia and bacteremia. On this basis, contemporary guidelines (ASCO/IDSA and ECIL) recommend fluoroquinolone prophylaxis for patients expected to experience profound neutropenia for > 7 days, a group that includes most recipients of myeloablative allo‐HCT in the peri‐transplant period. Nevertheless, the ECIL points out the lack of effect on overall mortality and that fluoroquinolone prophylaxis resulted in an increased colonization or infection with fluoroquinolone—or multi‐drug resistant strains [139, 140]. Therefore, the ECIL indicated that prophylaxis with fluoroquinolone should be weighed against its impact in terms of toxicity and changes in local ecology in single. Several clinical investigations have shown consistently that intensive gut decontamination with potent, non‐absorbable antibiotics such as rifaximin markedly reduces the risk of aGVHD after allo‐HCT [141, 142, 143, 144]. Interestingly, compared to patients receiving ciprofloxacin/metronidazole, patients on rifaximin showed lower 1‐year transplant‐related mortality (p = 0.04) and higher OS (p = 0.008).

Beside prophylactic antibiotics, most patients received broad‐spectrum antibiotics during allo‐HCT that profoundly disturb the beneficial intestinal microbiota, leading to a loss of commensal anaerobes and reduced microbial diversity [20]. Antibiotics, particularly targeting anaerobic bacteria, have been associated with an increased incidence of aGVHD as well as aGVHD‐related mortality [141]. Therefore, some researchers advocate the use of narrow‐spectrum antibiotics (e.g., cefepime or aztreonam), which may help preserve anaerobic species involved in maintaining an anti‐inflammatory gut environment [145, 146]. The timing of antibiotic administration is another critical consideration. Evidence suggests that post‐transplant antibiotic use, rather than pre‐transplant prophylaxis, may lower NRM [20]. Moreover, studies indicate that with close clinical monitoring and prompt therapeutic intervention upon suspicion of infection, outcomes can be not inferior to those achieved with routine prophylactic antibiotic use [147]. Taken together, infection prevention and control represent a key component of hematopoietic HCT management, with the core lying in the rational and judicious use of antibiotics to optimize patient outcomes.

3.2. Prebiotics, and Nutrition Support

Prebiotics are substrates that resist digestion by the host but can be metabolized by beneficial gut bacteria. The primary prebiotics are resistant starches, fructooligosaccharides (including inulin), and galactooligosaccharides. Within the gut, prebiotics are fermented to generate a variety of metabolites, such as SCFAs, which play critical roles in maintaining intestinal mucosal integrity and modulating host immune responses. Some studies have shown that resistant starch increases the abundance of butyrate and butyrate‐producing bacteria, thereby strengthening the intestinal barrier and improving outcomes in aGVHD [148]. Similarly, fructooligosaccharides have been reported to enhance gut microbial diversity, promote the proliferation of SCFA‐producing bacteria, and potentially expand Treg populations via microbiota modulation [98, 149]. In murine models, inulin has been demonstrated to improve gut microbial composition and preserve epithelial barrier function [150, 151], highlighting its potential as a dietary intervention for HCT recipients; a clinical trial is currently ongoing (NCT04111471). Moreover, oral administration of galactooligosaccharides has been shown in mice to mitigate lethal aGVHD [152], and corresponding clinical trials are also underway (NCT04373057).

Regarding patients' nutrition during neutropenic phase after allo‐HCT, low‐bacterial neutropenic diet remains the standard of care to minimize infection from ingested microbes. A large randomized noninferiority trial of a liberalized diet versus the neutropenic diet was recently conducted in patients undergoing intensive chemotherapy, autologous HCT or allo‐HCT [153]. Diet arm patients were encouraged to eat at least one daily serving of fresh fruits and/or vegetables and were permitted to eat pasteurized yogurt, while the neutropenic diet did not permit consumption of fresh fruits and vegetables. The trial was halted at the second interim analysis after enrolling 214 evaluable patients, because of the liberalized diet arm's major infection rate of 31.4%, surpassing the predefined stopping boundary, compared with 20.2% of patients in the ND arm. Furthermore, the caloric intake in the liberalized diet arm was not improved and there was no advantage in symptoms, quality of life, or survival. This study therefore indicate that liberalized diet seems not safe during the neutropenic phase after allo‐HCT and that alternative strategies are needed to improve patient nutrition without impairing safety.

Importantly, additional nutritional support can be provided either via enteral nutrition (EN) or parenteral nutrition (PN). Several studies have reported that EN, compared with PN, is associated with a lower incidence of aGVHD, including GI‐aGVHD, and reduced risk of infection [154, 155, 156, 157]. This benefit may be attributed to the direct digestion and absorption of nutrients through the gut, which promotes intestinal motility, preserves barrier function, and limits bacterial translocation. In contrast, prolonged PN can lead to mucosal atrophy and gut microbiota dysbiosis, thereby increasing susceptibility to infections [157, 158]. Finally, a pilot study compared outcomes between patients who received standard EN (n = 10) versus prebiotic fiber EN (n = 20) after allo‐HCT. There was no difference in clinical outcomes and microbial diversity declined in both groups; however, there was in the prebiotic groups an increase of Lactobacillus_C rhamnosus (p = 0.022) and a decrease in Faecalicatena gnavus (p = 0.0027) [159].

3.3. Probiotics, Including FMT

Probiotic strategies involve the supplementation of selected microbial strains—either single or a limited number, to modulate and support the gut microbiota. One study demonstrated that Lactobacillus brevis CD2 lozenges could prevent oral mucositis in patients undergoing HCT following high‐dose chemotherapy [160]. A pilot randomized clinical trial (n = 40) evaluated a commercially available symbiotic mixture containing high levels of 7 safe bacterial strains plus fructo‐oligosaccharides as a prebiotic administered 21 days prior to transplantation (Days −21 to Day 0) [98]. Use of symbiotic was associated with a reduced risk of severe grade III‐IV aGVHD and Tregs induction [98]. Another ongoing study (NCT03922035) found that administration of the probiotic formulation CBM588 during the peri‐transplant period was safe, modulated the gut microbiota, and provided early indications of reduced GI‐GVHD incidence and improved HCT‐related outcomes. Nevertheless, other reports have indicated that probiotic use may be associated with bacteremia [161, 162]. Importantly, these adverse events were linked to over‐the‐counter probiotic products rather than controlled therapeutic interventions. Accordingly, the clinical efficacy and safety of probiotic therapy in HCT patients should be interpreted with caution.

Compared with probiotic strategies, FMT delivers a complete and complex microbial community, enabling comprehensive remodeling of the gut microbiota [3, 163, 164, 165]. FMT has been widely demonstrated to be effective in treating Clostridioides difficile infections following transplantation [166, 167, 168]. It is also considered a safe and effective decolonization strategy for patients colonized or infected with multidrug‐resistant organisms [169]. Accumulating evidence indicates that FMT is safe and efficacious in managing steroid‐refractory and steroid‐dependent GI‐aGVHD populations [170, 171, 172]. Importantly, a recent pivotal phase III clinical trial (NCT04769895) reported that MaaT013, a microbiome ecosystem therapy administered via enema, that was first investigated in steroid‐refractory GI aGVHD [173], showed efficacy as a third‐line treatment for GI‐aGVHD [174]. In addition, DeFilipp et al. [175] investigated FMT combined with corticosteroids in patients with high‐risk lower GI‐aGVHD (n = 10), including 9 patients that were in first‐line treatment, with a CR rate at Day 28 of 70%, suggesting FMT may be a novel approach for first‐line treatment of aGVHD. Finally, FMT was also investigated in steroid‐refractory cGVHD (n = 12), with an ORR of 50% (1 CR and 5 PR) and an increased gut microbial diversity and higher abundance of SCFA‐producing bacteria in responding patients [176].

Besides treatment of GVHD, FMT was also investigated to restore gut microbiota diversity after intensive chemotherapy and use of broad‐spectrum antibiotics. First reports use autologous FMT, with stool collected and cryopreserved before start of the chemotherapy and delivered to the patients at neutrophil recovery after broad‐spectrum antibiotic discontinuation. Autologous FMT was associated with restoration of the gut microbiota diversity and composition [3, 163].

A double‐blind phase II study then randomized third‐party FMT versus placebo in 2 independent cohorts of allogeneic HCT recipients and patients with AML receiving induction chemotherapy [177]. In both AML and allo‐HCT groups, the primary endpoint, 4‐month all‐cause infection rate, was not reached, with no difference between the placebo and the FMT group. In the allo‐HCT group, a post hoc analysis suggests that FMT may have protective effects against aGVHD, especially in patients with more severe microbiota disruptions [165]. Another study randomized SER‐155, an investigational, oral live biotherapeutic comprised of 16 bacterial strains, versus placebo, after allo‐HCT and found a lower incidence of blood‐stream infection in SER‐155 versus placebo‐treated patients (10% vs. 42.9%, respectively) warranting further investigation [178]. Finally, MaaT033, a pooled, allogeneic, lyophilized, and standardized fecal microbiotherapeutic product, was shown to be safe and effective for gut microbiota restoration in patients with AML receiving induction chemotherapy [179] and is currently evaluated in a large phase IIb randomized placebo‐controlled study in allo‐HCT patients, with a primary endpoint on OS (NCT05762211).

Safety remains a central concern in immunocompromised patients receiving FMT, and several challenges persist, including the optimal route of administration, timing of transplantation, and ideal donor source. While studies have shown that FMT increases levels of butyrate and indole [180], whether additional biomarkers can reliably indicate therapeutic success requires further investigation.

3.4. Postbiotics

Postbiotic strategies directly deliver microbial metabolites, which may provide a safer therapeutic approach for immunocompromised recipients of allo‐HCT. As noted above, microbial metabolites such as SCFAs have demonstrated substantial therapeutic potential in transplant settings. In a murine allo‐bone marrow transplant model, exogenous butyrate supplementation restored intestinal epithelial cell junction integrity, reduced apoptosis, and attenuated GVHD [181]. Another study reported that administration of the indole derivative indole‐3‐carbaldehyde mitigated intestinal epithelial damage, limited bacterial translocation, and decreased pro‐inflammatory cytokine production, thereby reducing both GVHD incidence and associated mortality [182]. Additionally, a multicenter clinical study indicated that post‐allo‐HCT administration of ursodeoxycholic acid decreased pro‐inflammatory cytokine production and severe aGVHD, while improving overall patient survival [83, 183].

4. Conclusion and Future Directions

In this review, we have shown how the gut microbiota may maintain intestinal homeostasis and modulate the immune response after allo‐HCT, possibly contributing to improved clinical outcomes, underscoring its increasing clinical relevance. Of note, despite post‐transplantation cyclophosphamide being increasingly used as GVHD prophylaxis after allo‐HCT, not only for patients with a haploidentical donor, but also a matched related or unrelated donor [184], data regarding gut microbiome alterations in the setting of PTCy allo‐HCT are limited and future studies should investigate this question.

Given that the microbiota functions both as a biomarker and a therapeutic target, its dynamic alterations before and after allo‐HCT have substantial implications for patient management. Most compelling evidence regarding microbiota manipulation is in the setting of treatment of GI aGVHD, in particular with the positive pivotal phase III evaluating the microbiotherapy MaaT013 in steroid and ruxolitinib resistant GI aGVHD [174]. Nevertheless, additional data are needed to know if such treatment can be moved earlier in the history of the disease. Similarly, studies of prebiotics and synbiotics suggest potential benefits, but these findings still require validation in larger cohorts [148].

In addition, direct evidence is still lacking. Studies have identified associations between gut microbiota composition and transplant outcomes, including infection and survival; most interventional trials to date have primarily focused on aGVHD as the principal endpoint, with limited assessment of other clinically relevant outcomes, such as OS, NRM, and relapse. Further multicenter, standardized, and multi‐omics‐integrated studies are needed to determine whether modulation of the gut microbiota can truly improve overall post‐transplant outcomes.

In addition, existing studies are subject to multiple confounding factors. A study directly comparing autologous HCT and allo‐HCT suggested that antibiotic exposure, rather than the allogeneic response itself, is the major driver of post‐transplant microbiome injury [185]. Conditioning regimen is another important confounder: a study including 1188 allo‐HCT patients showed that different conditioning regimens were associated with distinct gut microbiome patterns [4]. Moreover, donor‐ and transplant‐related variables themselves have a substantial impact on clinical outcomes. HLA mismatch, age, GVHD prophylaxis regimen, and female donor‐to‐male recipient transplantation are all risk factors [186, 187, 188]. Therefore, analyses that do not adequately adjust for these variables are highly susceptible to confounding, and many reported associations may be overestimated or misinterpreted.

Therefore, the standardization of microbiome assessment methodologies is required to ensure data comparability and reproducibility across studies, thereby facilitating the translation of microbiome‐based discoveries into broader clinical practice. At the same time, ethical and privacy considerations must be carefully addressed, as microbiome data may contain sensitive personal information. Establishing a comprehensive ethical and regulatory framework, including informed consent, data security, and responsible commercialization, is therefore essential to safeguard patient rights and promote responsible research. Finally, improving the cost‐effectiveness and clinical applicability of microbiome detection technologies will be essential for promoting their clinical translation and broader implementation.

Funding

The authors have nothing to report.

Ethics Statement

The authors have nothing to report.

Conflicts of Interest

F.M. reports lecture honoraria from Amgen, Therakos/Mallinckrodt, BMS, MSD, Sanofi, Novartis, Astra Zeneca, and JAZZ Pharmaceuticals, all outside the submitted work. N.S. reports lecture honoraria from AbbVie, Astra Zeneca, BeOne Medicines, Johnson and Johnson Innovative Medicine, Lilly, and Novartis. M.M. reports grants and/or lecture honoraria from Janssen, Sanofi, Maat Pharma, JAZZ Pharmaceuticals, Celgene, Amgen, BMS, Takeda, Pfizer, Novartis, and Roche, all outside the submitted work. The other authors declare no conflicts of interest.

Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.