Intestinal reengineering: Scientific advances in intestinal transplantation

Abstract

Intestinal transplantation (ITx) has emerged as a pivotal life-saving intervention for patients with irreversible intestinal failure unresponsive to conventional medical and nutritional therapies. Despite its growing clinical acceptance, ITx remains among the most immunologically complex and technically demanding procedures in the field of solid organ transplantation. This review comprehensively summarizes the historical evolution, clinical indications, and advancements in surgical techniques, with emphasis on innovations in vascular anastomosis, multivisceral transplantation, and ex vivo preservation. Special attention is given to the unique immunological challenges of ITx, including bidirectional immune responses-host-vs-graft and graft-vs-host disease-immune-microbiota interactions, and the distinct roles of key immune cells. Pediatric and adult recipients exhibit divergent etiologies, immune responses, and complication profiles, necessitating individualized approaches. Although novel immunotherapeutic strategies and bioengineering innovations have improved short-term outcomes, chronic rejection, graft dysfunction, and immunosuppressive toxicity remain significant barriers. Looking ahead, future directions should prioritize precision immunomodulation, microbiome-targeted therapies, and integrated platforms for gene editing, 3D bioprinting, and immune monitoring. Through multidisciplinary collaboration and translational research, ITx is poised to evolve from a high-risk salvage therapy into a personalized, sustainable solution that enhances long-term survival and patient quality of life.

Key Words: Intestinal transplantation; Immune rejection; Intestinal flora; Immunosuppression; Intestinal autotransplantation

Core Tip: This review systematically elucidates the current status and challenges of intestinal transplantation (ITx) as a life-saving therapy for patients with irreversible intestinal failure. Despite therapeutic hurdles such as the high immunogenicity of the intestine, complex microbiota microenvironment, and susceptibility to rejection, surgical innovations-including robot-assisted vascular anastomosis and multivisceral transplantation-along with breakthroughs in ex vivo perfusion technology have significantly improved clinical outcomes. This review highlights that bidirectional immune reactions (host-vs-graft response and graft-vs-host disease) and chronic rejection remain the primary clinical obstacles. Analysis herein reveals marked differences in immunological profiles and clinical manifestations between pediatric and adult recipients, necessitating individualized treatment strategies. Based on current research advances, future directions include precision immunotherapy, gut microbiota modulation, and bioengineering innovations such as 3D bioprinted grafts. The review emphasizes that only through multidisciplinary collaboration can ITx evolve into a safer and more effective treatment, ultimately achieving comprehensive improvements in long-term survival rates and quality of life for patients.

INTRODUCTION

The small intestine is one of the largest immune organs in the human body and plays a critical role in maintaining health, sustaining life, and improving patients’ quality of life[1]. Over the past few decades, organ transplantation has achieved remarkable success in the cardiovascular, gastrointestinal, and urologic systems, with increasingly refined techniques saving the lives of countless patients. Despite persistent challenges-including donor shortages, immune rejection, and complexities in long-term postoperative management-these procedures have significantly advanced human health. Compared with established procedures such as liver, kidney, and heart transplantation, intestinal transplantation (ITx) remains a relatively recent advancement. Although improvements in surgical techniques and immunosuppressive therapies have facilitated progress, ITx continues to face considerable barriers, particularly high rates of graft rejection and postoperative complications, which limit its broader clinical application[2]. The small intestine is densely populated with lymphoid tissue within the mucosal layer, and hosts a vast array of microorganisms in the lumen, making it a highly immunogenic organ and a critical site of mucosal immune activity[3]. Immune interactions between host tissues and microbial communities can trigger infections and provoke intense immune responses post-transplantation, including host-vs-graft reaction (HVGR) and graft-vs-host reaction (GVHR)[4]. Moreover, many ITx candidates are immunocompromised due to long-term dependence on parenteral nutrition (PN), creating a dual challenge of immune deficiency and heightened rejection risk, both of which contribute to reduced surgical success rates[5,6].

In this article, we provide a comprehensive review of the historical evolution and recent breakthroughs in ITx surgical techniques, examine its unique immunological features and underlying mechanisms, and explore current clinical challenges and future directions. Our goal is to offer a theoretical foundation and practical strategies to support the successful clinical translation and broader application of ITx.

ITX: HISTORY AND CURRENT STATUS

The development of ITx has progressed over six decades, marked by key innovations in immunosuppression, surgical technique, and organ preservation. Initial attempts in the 1960s, including those by Lillehei and colleagues, failed due to inadequate immunologic control[7-9]. The introduction of total PN (TPN) in the 1980s temporarily reduced the urgency for ITx[10].

A major breakthrough occurred in the early 1990s with the clinical application of tacrolimus, which significantly improved one-year graft survival and established ITx as a viable treatment option for irreversible intestinal failure[11]. Since then, the field has entered a period of steady advancement, including the development of multivisceral transplantation protocols, improved immunosuppressive regimens, and organ preservation strategies such as normothermic machine perfusion (NMP) and ex vivo normothermic perfusion (EVNP)[6,12,13]. More recent innovations include: 3D-bioprinted vascularized intestinal scaffolds, offering future solutions to organ shortages[14,15]; Precision immunomodulatory strategies, such as Janus kinase (JAK) inhibitors and chimerism-based tolerance[16,17]; Microbiome-targeted therapies, including personalized fecal microbiota transplantation (FMT)[18]. Figure 1 presents a chronological timeline of these technological and clinical milestones, contextualizing the evolution of ITx alongside persistent challenges that continue to shape the field.

Figure 1 The historical development and challenges of intestinal transplantation. TPN: Total parenteral nutrition; ITx: Intestinal transplantation; CR: Chronic rejection; AMR: Antibody-mediated rejection; FMT: Fecal microbiota transplantation; JAK: Janus kinase.

TRANSITION FROM INTESTINAL FAILURE TO TRANSPLANT CANDIDACY

Etiologies of intestinal failure

Intestinal failure is defined as the inability of the gut to absorb sufficient nutrients, fluids, and electrolytes to sustain growth, development, or homeostasis without parenteral support. It can result from either anatomical loss or functional impairment of the intestine. Anatomical causes include short bowel syndrome (SBS) due to massive small bowel resection, mesenteric ischemia, trauma, congenital intestinal atresia, or enterocutaneous fistulas. Functional causes include disorders such as chronic intestinal pseudo-obstruction, severe motility disorders, mucosal diseases (e.g., microvillus inclusion disease), and radiation enteritis[19,20]. These conditions may be congenital or acquired and can affect both children and adults.

Indications for ITx

Although PN remains the cornerstone of treatment for patients with intestinal failure, long-term PN dependence is associated with significant morbidity, including catheter-related bloodstream infections, metabolic bone disease, loss of venous access, and intestinal failure-associated liver disease (IFALD). ITx is not considered a first-line or standard therapy for intestinal failure but is instead reserved for carefully selected patients who either develop life-threatening complications from PN or in whom intestinal rehabilitation has failed to achieve nutritional autonomy[21]. The internationally accepted clinical indications for ITx include: (1) Irreversible IFALD with evidence of progressive liver dysfunction; (2) Recurrent catheter-related sepsis despite preventive strategies; (3) Loss of central venous access due to thrombosis; (4) Fluid and electrolyte imbalances unresponsive to optimized PN; and (5) Severe growth failure or poor quality of life despite maximal medical and nutritional support[21,22]. These indications reflect the transition from PN failure to transplant candidacy and highlight the role of ITx as a critical therapeutic intervention when conventional strategies are exhausted. The decision to proceed with transplantation should be made within the framework of a multidisciplinary intestinal rehabilitation program, incorporating surgical, nutritional, infectious disease, and psychosocial expertise to ensure optimal patient selection and timing[23].

Importantly, with advances in multidisciplinary rehabilitation, the threshold for proceeding to ITx has risen, and many patients who would previously have been considered transplant candidates can now be successfully managed with integrated medical-nutritional approaches. As such, ITx remains a vital but highly specialized therapy, reserved for a minority of patients with refractory or complicated intestinal failure.

CHARACTERISTIC DIFFERENCES IN ITX BETWEEN PEDIATRIC AND ADULT HUMANS

Since its clinical introduction in the 1980s, ITx has substantially improved both survival rates and quality of life for patients dependent on PN. While allogeneic transplantation shares fundamental immunological and surgical principles across age groups, pediatric and adult recipients differ significantly in terms of etiology, anatomical and physiological characteristics, perioperative management, and long-term outcomes. These differences necessitate tailored transplantation strategies and contribute to distinct clinical trajectories. Data from the International Intestinal Transplant Registry (IITR) indicate that adult ITx recipients have 5-year survival rates of approximately 55%-65%, whereas pediatric recipients demonstrate slightly better outcomes, with 5-year survival ranging from 60%-70%. However, pediatric ITx is associated with unique challenges, including limited donor availability, the need for long-term surveillance of growth and neurodevelopment, and the importance of sustained family and psychosocial support[24]. In contrast, adult recipients often present with more complex underlying conditions or comorbidities at the time of transplantation. Although combined liver-ITx has been shown to reduce acute rejection (AR) rates in adults, it adds surgical complexity and may increase the risk of postoperative infections and complications[25].

Currently, most studies focus on either pediatric or adult ITx cohorts in isolation, with limited systematic comparisons between the two groups. Addressing this gap through well-designed comparative studies represents an important future direction for advancing individualized care and optimizing outcomes across the lifespan.

Etiological differences

The differences between pediatric and adult ITx primarily arise from distinct etiologies and clinical indications. In pediatric patients, intestinal failure is most commonly caused by congenital or perinatal conditions, such as intestinal atresia or necrotizing enterocolitis (NEC), which may result in intestinal dysfunction or SBS following surgical resection. Additionally, prolonged dependence on PN in pediatric SBS frequently leads to IFALD-a term now preferred over the outdated “PN-associated liver disease”-to better reflect its multifactorial pathophysiology involving PN, recurrent sepsis, and underlying intestinal compromise. The development of IFALD in pediatric patients is a major trigger for early transplant consideration, aiming to prevent irreversible progression to end-stage liver disease[26,27]. In contrast, adult ITx candidates more often present with acquired conditions such as mesenteric ischemia, Crohn’s disease, post-traumatic intestinal loss, or long-term PN-related complications. The primary therapeutic goals in adults typically emphasize achieving PN independence and restoring intestinal function[20,28].

Another critical factor contributing to the divergence in pediatric vs adult ITx lies in anatomical and physiological differences, which directly impact surgical complexity, postoperative recovery, and long-term outcomes. Pediatric intestines are approximately six times the length of the body-often exceeding adult intestinal length-highlighting key anatomic distinctions[29,30]. Furthermore, the pediatric mesenteric vasculature and intestinal lumen are markedly narrower, increasing technical demands during transplantation. This necessitates highly precise vascular anastomosis of major structures such as the portal vein and mesenteric arteries. Nevertheless, the relatively longer intestinal length in pediatric patients may also confer superior regenerative capacity compared to adults.

Immunological differences

From an immunological standpoint, pediatric ITx recipients exhibit a higher risk of AR compared to adults, primarily due to their immature immune systems and limited immunocompetence. This necessitates the use of more precise and personalized immunosuppressive regimens for pediatric patients. Nevertheless, pediatric recipients generally demonstrate superior graft survival outcomes relative to adults, which is attributed to distinct immunobiological characteristics and customized clinical management strategies. Although children are more susceptible to AR, their immune systems demonstrate notable plasticity and adaptability. Recipients under the age of five exhibit increased frequencies of peripheral regulatory T cells, enhanced thymic output, and heightened sensitivity to calcineurin inhibitors[24,31]. While living-related intestinal donation has been attempted in select pediatric cases, the vast majority of both pediatric and adult ITxs are conducted using deceased donors[32]. Therefore, the assumption that pediatric recipients commonly receive grafts from related living donors does not reflect current clinical practice. Strategies to minimize HLA mismatch and improve graft outcomes are implemented in both populations, irrespective of donor source[33]. In clinical management, pediatric protocols often involve more frequent immunological monitoring and earlier therapeutic interventions, which contribute to higher rates of rejection reversal[34]. Although infectious complications are more commonly observed more frequently in children, these events less often lead to graft loss[35].

Collectively, these factors establish a unique “high-responsiveness/high-controllability” immunological paradigm in pediatric recipients, ultimately contributing to their superior long-term graft survival. In contrast, adult recipients are more prone to antibody-mediated rejection (AMR) and chronic graft dysfunction, likely due to a greater burden of preformed antibodies and memory T cells[36], underscoring the fundamental immunological differences between the two groups. The development of combined liver-ITx and refinement of immunosuppressive strategies have helped mitigate rejection risks in adults, a protective effect thought to arise from the liver’s intrinsic immunomodulatory capacity[37]. This protective effect is believed to play a role in attenuating intestinal graft rejection. Both adult and pediatric recipients are vulnerable to complications arising from prolonged immunosuppressive therapy, although their risk profiles vary significantly. Adults are more likely to develop metabolic and cardiovascular side effects-such as post-transplant diabetes mellitus, hypertension, and osteoporosis-often worsened by preexisting comorbidities and age-related organ decline[38]. In contrast, pediatric patients, owing to their longer life expectancy and continued growth, face cumulative toxicities, including growth retardation, nephrotoxicity, neurocognitive effects, and increased infection risk[39]. Therefore, the severity and manifestation of immunosuppression-related complications are not necessarily greater in one population, but rather reflect distinct physiological and temporal vulnerabilities, necessitating tailored long-term management strategies[40].

Currently, immune induction regimens differ slightly between pediatric and adult ITx patients. Pediatric protocols typically include rATG, alemtuzumab (used increasingly in recent practice), basiliximab, and rituximab (used less frequently over time). The reported rates of no rejection, moderate rejection, and severe rejection in the perioperative period are approximately 65%, 22%, and 13%, respectively. In adults, commonly used induction agents include rATG (gradually tapered in clinical use), basiliximab (increasingly utilized), alemtuzumab (gradually reduced), and rituximab (used more frequently in recent protocols). The corresponding rates of no rejection, moderate rejection, and severe rejection in adults are 79%, 15%, and 6%, respectively. Maintenance immunosuppressive regimens are generally similar across both populations and include tacrolimus (91%), corticosteroids (55%), sirolimus (16%), mycophenolate mofetil (12%), azathioprine (6%), cyclosporine (3%), and other agents (4%).

Differences in long-term survival rate and complications

Data from the IITR and recent clinical studies indicate that pediatric ITx recipients generally achieve higher 5-year survival and graft survival rates compared to adult recipients[41]. This favorable outcome is attributed to immunological adaptability, better regenerative capacity, and improved perioperative management in pediatric patients. Although children are more prone to early infectious complications, they exhibit higher rates of rejection reversal and demonstrate more durable long-term graft function[42].

In pediatric ITx recipients, the rank order of graft survival duration is as follows: Total abdominal organ cluster transplantation > combined liver-ITx > isolated small bowel transplantation > modified abdominal cluster transplantation. Conversely, for adult recipients, the order differs slightly: Combined liver-ITx > modified abdominal cluster transplantation > isolated small bowel transplantation > total abdominal organ cluster transplantation.

The incidence of long-term complications also varies between pediatric and adult populations. Post-transplant insulin resistance occurs in approximately 1.8% of pediatric patients compared to 7% in adults; hypertension in 9% of children vs 16% in adults; continuous renal replacement therapy is required in 3% of pediatric vs 6% of adult recipients; and the incidence of post-transplant lymphoproliferative disorder (PTLD) is 8% in children compared to 4% in adults. These findings highlight age-related disparities in complication profiles, likely reflecting differences in baseline comorbidities, immune status, and immunosuppressant pharmacokinetics.

In conclusion, the fundamental differences in ITx outcomes between pediatric and adult patients arise from a complex interplay of etiological, anatomical, immunological, and psychosocial factors. Future efforts should focus on optimizing donor-recipient matching in pediatric ITx, designing immunosuppressive regimens with reduced toxicity, and strengthening multidisciplinary post-transplant care to improve long-term prognosis.

CLINICAL APPLICATION OF ITX SURGERY

ITx represents the most technically demanding procedure among abdominal organ transplants, requiring precise surgical decision-making to balance the extent of bowel resection during the initial operation. The goal is to avoid excessive resection that may precipitate intestinal failure, while also minimizing complications associated with retaining dysfunctional or marginal bowel segments[43]. In pediatric patients, assessment of residual bowel length must be made in relation to age-specific norms. Early stoma closure is often considered, and in some cases, this is complemented by proximal effluent refeeding to promote intestinal adaptation and enteral autonomy[44]. When significant bowel dilation is present, reconstructive techniques such as serial transverse enteroplasty (STEP) may be employed to improve functional outcomes. In contrast, adult patients often respond adequately to simpler bowel tapering procedures, which are effective in managing dilated segments and enhancing nutrient transit[31,45]. In refractory cases, comprehensive surgical and functional evaluation is essential to determine whether reconstructive approaches alone are sufficient or whether ITx is warranted.

Recent advances in multivisceral transplantation techniques and refined vascular anastomosis methods have led to notable improvements in 5-year survival rates among adult ITx recipients. However, critical challenges persist, including donor organ shortages, size mismatches, and the ongoing risk of chronic rejection (CR). Ultimately, successful ITx outcomes depend on individualized surgical planning that incorporates multiple patient-specific factors, including age, anatomical variability, underlying pathology, and residual intestinal function-fully aligning with the principles of precision medicine.

Breakthroughs in ITx vascular anastomosis technology

Vascular anastomosis forms the cornerstone of many modern surgical procedures and has undergone continuous refinement in the context of ITx. In early ITx animal experiments, even when vascular continuity was successfully re-established through precise vessel suturing, graft loss frequently occurred due to uncontrolled immune rejection. This highlighted that vascular anastomosis alone could not ensure graft survival, and only after the introduction of effective immunosuppressive agents did ITx gain momentum, enabling meaningful advances in vascular reconstruction techniques[7]. Traditional ITx involves direct anastomosis of the donor mesenteric artery to the recipient’s abdominal aorta and inferior vena cava. However, this approach is technically challenging due to the depth and short length of the native vessels, which increases the risk of thrombosis and prolongs operative time. To overcome these challenges, the "vascular bridge" technique was developed, allowing the use of donor iliac vessels or synthetic grafts to convert complex end-to-side anastomoses into more manageable end-to-end configurations, thereby reducing operative time and vascular complications[46].

In complex surgical scenarios-such as patients with traumatic abdominal wall defects, multiple cesarean deliveries, or extensive intestinal fistulas leading to loss of abdominal domain and severe scarring-reconstruction of the abdominal wall may require abdominal wall vascularized composite allotransplantation. This approach is often performed in conjunction with ITx or multivisceral transplantation, and in these cases, vascular anastomosis and revascularization are pivotal to procedural success[47]. Therefore, techniques for vascular anastomosis and revascularization in this combined transplantation context are central to achieving optimal outcomes. Levi et al[48] pioneered the use of end-to-end anastomosis between the donor infra-abdominal vessels (including iliofemoral segments) and the recipient’s common iliac vessels. While this technique offered a macroscopically accessible anastomotic site, it did not allow simultaneous perfusion of the graft, leading to prolonged ischemia time.

In a related innovation, small bowel autotransplantation has been introduced as a potential option for the resection of tumors involving the mesenteric vasculature. This technique allows radical tumor removal while maintaining bowel viability and intestinal continuity[49]. Cipriani et al[50] proposed a microvascular end-to-end anastomosis technique between the donor and recipient infra-abdominal vessels, while preserving the donor’s iliofemoral vasculature. This strategy enhanced vascular control and reduced donor morbidity. Later, Giele et al[51] subsequently introduced a two-stage approach, initially anastomosing the graft’s infra-abdominal vessels to the recipient’s forearm vessels to allow early graft perfusion and stabilization. The graft was then transferred to the abdominal cavity after recipient stabilization. More recently, Erdmann et al[52] described a technique enabling simultaneous hemodialysis by constructing a vascular ring between the saphenous vein and the common femoral artery. However, this method is unsuitable in cases of prior saphenous vein injury or thrombosis.

Potential advantages of robotic-assisted vascular anastomosis in ITx

Robotic-assisted surgery has demonstrated considerable advantages in urological operations, including partial and radical nephrectomy, due to its enhanced three-dimensional visualization, refined instrument articulation, and significant reduction in intraoperative blood loss[53]. Importantly, growing evidence supports robotic-assisted kidney transplantation as a feasible and often superior alternative to open surgery, with reduced postoperative complications and improved surgical precision. This technique proves particularly beneficial during critical steps such as vascular anastomosis and ureteral reconstruction, especially in patients with obesity or complex anatomical variations[54,55].

In the domain of ITx, vascular anastomosis-especially involving the superior mesenteric artery (SMA)-presents substantial technical challenges. Adequate blood perfusion must be ensured while minimizing ischemia-induced graft injury[56]. Although conventional open surgery remains the clinical standard for SMA reconstruction, robotic-assisted platforms offer promising solutions through magnified visualization, tremor suppression, and high-precision instrument control. The integration of intraoperative fluorescence imaging, such as indocyanine green, enables real-time assessment of mesenteric perfusion, thereby reducing the likelihood of anastomotic failure[57]. Furthermore, robotic systems provide enhanced dexterity for complex vascular procedures-including end-to-end microanastomosis, venous patching, and customized bypass grafting-potentially reducing operative time and improving surgical outcomes in ITx recipients[58]. These capabilities may be particularly valuable in reoperations, pediatric cases, or recipients with prior abdominal interventions.

While clinical reports on robotic-assisted ITx remain limited, the theoretical and technological potential is substantial. Future directions in this field should prioritize: (1) Establishing standardized robotic-assisted protocols tailored to the anatomic and hemodynamic features of intestinal graft vasculature, such as mesenteric artery and vein variants; (2) Conducting multicenter prospective trials to evaluate long-term graft survival, immunologic outcomes, and complication rates in robotic-assisted ITx; (3) Fostering interdisciplinary surgical integration, uniting transplant surgeons, vascular specialists, and robotic experts to optimize perioperative workflows and minimize conversion rates; and (4) Developing next-generation materials and robotic-compatible vascular scaffolds, including anti-adhesive bioabsorbable stents and microvascular sealants specifically designed for intestinal and mesenteric applications. Collectively, these strategies hold promise to transform robotic-assisted ITx from an emerging concept into a reliable and innovative surgical paradigm, enabling safer, more precise, and personalized management of complex intestinal failure.

Technical advances in combined multi-organ ITx

Allogeneic ITx remains the most widely used modality for treating isolated small bowel failure. For patients with preserved function of other digestive organs, isolated jejunal or ileal ITx is sufficient to restore intestinal continuity and absorption. However, in cases of multi-organ dysfunction-such as coexisting liver failure, pancreatic tumors, or complex hepatobiliary disease-isolated ITx often proves inadequate, prompting the clinical adoption of combined organ transplantation, including liver-small bowel or multivisceral (e.g., small intestine, pancreas, stomach) transplantation[59,60]. Among multivisceral transplant options, liver-small bowel transplantation is most commonly performed, with the liver playing a central immunologic and metabolic role. However, organ selection must be individualized based on the patient's pathology. For example, patients with mild hepatic dysfunction (e.g., early-stage cholestasis) without significant cirrhosis or portal hypertension may be better served by isolated ITx, preserving native liver function and its immunomodulatory benefits[25,61]. Moreover, isolated ITx is associated with lower surgical trauma and reduced thrombotic risk, compared to the more complex vascular reconstructions required in combined liver transplantations. Conversely, for patients with advanced liver disease secondary to IFALD, combined liver-small bowel transplantation remains indispensable. Liver dysfunction in PN-dependent patients can be assessed through serum biomarkers-such as albumin, bilirubin, and prothrombin time-which correlate with disease severity and the need for combined transplantation[62]. When performing combined hepatic-ITx, the pancreaticoduodenal arc is often transplanted en bloc with the liver to preserve structural and vascular integrity, reducing operative complexity. However, this approach also increases technical demands, requiring simultaneous reconstruction of the hepatic artery, portal vein, and biliary system, as well as high-level immunological management[63].

In complex digestive reconstructions, extended graft combinations-encompassing stomach, duodenum, pancreas, bile duct, colon, and spleen-are utilized by select transplant centers. These components can be harvested en bloc or as modular grafts depending on donor anatomy, allowing preservation of vascular and neural continuity[64-66]. This method is often referred to as abdominal organ cluster transplantation or modified cluster transplantation, and organ selection should be tailored to the patient's anatomical limitations and risk profile. Despite growing use, regional and institutional variability persists, and further empirical studies are needed to evaluate the survival benefit of various combinations.

A key area of innovation lies in colon inclusion within the ITx graft. Historically, concerns about increased infection risk and graft failure led to reluctance in including the colon. However, recent data demonstrate that colon co-transplantation does not increase infection or mortality rates, and may confer significant advantages. Specifically, colon recipients show improved fluid retention, less dehydration, and better renal outcomes due to increased fecal water reabsorption. Currently, colon inclusion is reported in nearly 20% of ITx cases[67,68]. The inclusion of the spleen remains controversial. On one hand, preserving the recipient spleen may reduce the risk of PTLD[69]. On the other hand, co-transplantation of the donor spleen in multivisceral ITx has been shown to promote immune tolerance and reduce AR, without increasing the incidence of graft-vs-host disease (GVHD)[65,70]. Strategies for gastric graft inclusion also vary widely across transplant centers. Some incorporate the stomach to preserve vagal nerve integrity and enhance neuromodulatory function of the graft, while others avoid gastric inclusion due to uncertain benefits and potential risks. Current evidence is insufficient to establish standardized recommendations, and future research should focus on evidence-based risk-benefit assessment of gastric co-transplantation[66].

Synergistic optimization of ischemia-reperfusion injury and ex vivo perfusion technology

Ischemia-reperfusion injury (IRI) remains a major limiting factor in ITx, not only contributing directly to graft dysfunction and rejection but also significantly constraining the effective duration of ex vivo preservation. Unlike other solid organs that can be preserved for over 24 hours, small intestinal grafts are far more sensitive to IRI, with current EVNP systems maintaining viability for only 8-12 hours[13]. This limitation arises from the intestine's highly vascularized mucosa and its complex immune-microbial interface, which together initiate a cascade of pathophysiological events during IRI: (1) ATP depletion during ischemia, followed by reperfusion, leads to mitochondrial dysfunction and bursts of reactive oxygen species (ROS), primarily through NADPH oxidase and xanthine oxidase activity, resulting in direct epithelial injury[71]; (2) Gut dysbiosis, especially overgrowth of opportunistic bacteria such as Escherichia coli, triggers systemic inflammation via Toll-like receptor (TLR)4/NF-κB signaling, elevating interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) levels and compounding IRI-induced tissue damage[72]; and (3) IRI-induced upregulation of HLA class I and II on epithelial cells primes the immune system by enhancing donor-specific antibody (DSA) formation and T-cell-mediated responses, thereby increasing the risk of AR[73,74].

Recent developments in machine perfusion have shown that NMP significantly outperforms hypothermic machine perfusion in preserving intestinal grafts. NMP maintains physiological conditions, preserves mucosal architecture and microcirculatory integrity, and reduces epithelial disruption, thereby prolonging viable preservation times[12]. Experimental porcine models of EVNP have been successfully established, allowing for real-time evaluation of intestinal viability and perfusion parameters, bridging the gap between bench and bedside[75,76]. Concurrently, the emergence of 3D-bioprinting technologies has enabled the creation of bioengineered intestinal scaffolds using light-polymerized hydrogels that mimic villus-crypt architecture and incorporate prevascularized networks. These constructs show promise in addressing post-transplant no-reflow phenomena by facilitating rapid graft revascularization[14,15].

On the pharmacological front, several novel interventions are being integrated into EVNP protocols to enhance graft resilience. Tranilast, for instance, upregulates heme oxygenase-1 (HO-1), whose metabolic product-carbon monoxide-inhibits mitochondrial permeability transition pore opening, thereby reducing mucosal apoptosis and increasing ischemia tolerance in preclinical settings[72,77]. Combinatorial approaches are also gaining traction. For example, mitochondrial-targeted antioxidants such as MitoQ scavenge ROS to preserve ATP synthesis and mitochondrial membrane potential[78], while probiotic strains like Clostridium butyricum help maintain microbial homeostasis through competitive exclusion of pathogens and secretion of short-chain fatty acids (SCFAs), collectively reducing IRI severity and extending ex vivo preservation limits[79].

The convergence of machine perfusion, tissue engineering, and molecular pharmacology marks a transformative phase for ITx. Future research should focus on refining EVNP protocols compatible with multi-organ preservation platforms to extend graft viability beyond 24 hours, validating 3D-bioprinted vascularized scaffolds in large-animal transplantation models, and designing synergistic therapeutic regimens that combine HO-1 inducers with microbiota-targeting strategies to promote immune quiescence and graft adaptability. These integrative approaches are expected to expand the donor organ pool, alleviate post-transplant immunological stress, and accelerate the development of next-generation ITx grafts characterized by enhanced immunotolerance and functional resilience.

IMMUNOLOGICAL ADVANCES IN ITX

Fundamental immunological characteristics of ITx

ITx remains a vital intervention for irreversible intestinal failure; however, its distinct immunological complexity renders it the most rejection-prone abdominal organ transplant. This high immunogenicity stems from the small intestine’s unique structural and immune properties, including its vast mucosal surface, rich lymphoid architecture, and dense commensal microbiota-factors that collectively heighten the risks of graft rejection and infectious complications[80].

The immune microenvironment of the small intestine comprises specialized immune-active compartments such as lamina propria lymphocytes, Peyer’s patches, and mesenteric lymph nodes[81]. Following ITx, the recipient’s immune system detects donor-derived alloantigens-primarily mismatched HLA molecules-activating both adaptive and innate immune pathways. This immunological cascade culminates in graft-directed immune injury, presenting clinically as AR or CR[82]. HVGR is driven by the activation of recipient T and B lymphocytes upon recognition of foreign HLA, initiating cytotoxic and antibody-mediated mechanisms of graft injury. The incidence of AR in ITx is reported at 30%-50%, which is significantly higher than that observed in other solid organ transplants such as the liver or kidney, and untreated CR often leads to progressive fibrosis and irreversible graft dysfunction[83,84].

In contrast to other abdominal organ transplants, ITx is uniquely susceptible to GVHD, in which immunocompetent donor-derived T cells recognize host tissues as foreign and mount a systemic immune attack. GVHD in ITx primarily targets recipient skin, liver, and gastrointestinal tract, often manifesting as severe inflammatory injury[85]. Although its pathogenesis remains incompletely understood, recent evidence implicates the activation of innate immunity via pathogen-associated molecular patterns (PAMPs), mucolytic bacterial disruption of the epithelial barrier, and dysregulated host metabolite signaling as key contributors to the onset and severity of GVHD[86]. Both acute and chronic GVHD represent major causes of post-ITx morbidity and mortality[87], with incidence rates exceeding those seen in liver and kidney transplants due to the high immunological load of the intestinal graft[88]. The risk and severity of GVHD are closely linked to donor-recipient HLA mismatch and the intensity of immunosuppression. Preventive strategies under investigation include pre-transplant lymphocyte depletion, donor bone marrow infusion, and early modulation of innate immune signaling[89].

Cellular mechanisms of rejection and tolerance

Following ITx, the transplanted intestine serves both as a frontline defense against external antigens and a persistent source of donor-derived alloantigens, rendering it highly immunogenic and especially vulnerable to rejection compared to other solid organ transplants[90]. Immune cells play a pivotal role in this delicate immune balance-orchestrating both AR and CR processes, while also mediating immune tolerance through regulatory pathways. Table 1 outlines the primary immunological cell types involved in ITx and their associated mechanisms. A comprehensive understanding of the dual roles of immune cells-in mediating rejection and promoting tolerance-is essential for optimizing immunosuppressive regimens and developing next-generation immune-modulating therapies.

Table 1 Potential functions and mechanisms of immune cells in small intestine transplantation.

Immune cell type	Infiltration characteristics and functions	Related mechanisms/molecular markers	Clinical significance	Ref.
T cell (CD8+)	Key effector cells in early AR; may develop exhausted-like phenotype under persistent antigen stimulation	PD-1, TIM-3, IFN-γ	AR biomarker; PD-1 inhibition must consider graft-vs-host disease risk	[209-211]
T cell (CD4+)	Th1/Th17 subsets drive rejection; Treg maintains mucosal homeostasis by suppressing inflammation	Th17: IL-17; Treg: FoxP3, IL-10, TGF-β	Th17/Treg ratio reflects rejection risk; modulating this axis improves outcomes	[91,92,212]
Regulatory T cells (Tregs)	Inhibit effector T cell activation; key regulatory cells promoting transplant tolerance	FoxP3, CTLA-4, IL-10, TGF-β	Treg expansion strategies may enhance tolerance and reduce rejection	[104,105,213]
B cell	Mediate chronic rejection and vascular injury via DSA production	IgG/IgM, complement activation, intimal hyperplasia	DSA levels correlate with chronic rejection; rituximab as therapeutic option	[164,214,215]
Natural Killer Cell (NK)	Contribute to rejection via ADCC; low-activity NK may support tolerance	NKG2D, perforin/granzyme, IL-10 inhibition	Regulating NK may reduce antibody-mediated rejection and mucosal injury	[101,216,217]
DC	Activate T cells in lymph nodes; tolerogenic DCs induce Treg differentiation	cDC1/cDC2 antigen presentation; pDC: IFN-α; tolerogenic DC: PD-L1	DC subpopulation imbalance promotes Th1 polarization; targeting PD-L1 can enhance transplant tolerance	[218-220]
Macrophage	M1 aggravates ischemia reperfusion injury; M2 promotes tissue repair and immune suppression	M1: TNF-α, IL-6; M2: ARG1	M1/M2 balance affects prognosis and barrier preservation	[221-224]
Mast cell	Disrupt mucosal barrier; associated with anastomotic leakage and infection	Histamine, IL-4, IL-13, eosinophil recruitment	Antihistamines or stabilizers may prevent complications	[225,226]
Myeloid inhibitory cells (MDSC)	Suppress T cell responses; excessive accumulation may lead to fibrosis	ARG1, ROS, M-MDSC	Targeted MDSC modulation needed to avoid immune imbalance	[227-229]
γδT cell	Regulate epithelial repair and microbial homeostasis; produce IL-17 and IL-22	γδT17 subset, IL-17, IL-22, villous regeneration	Support mucosal regeneration; crucial in post-ITx repair	[230,231]
ILC	ILC3 preserve epithelial integrity via IL-22; ILC2 mediate type 2 responses and may influence fibrosis	ILC3: RORγt, IL-22; ILC2: IL-5, IL-13	Dysfunction linked to chronic inflammation; IL-22 as repair target	[232,233]

AR: Acute rejection; PD-1: Programmed cell death 1; IFN: Interferon; DSA: Donor-specific antibody; DC: Dendritic cells; ILC: Innate lymphoid cells.

Pro-inflammatory effector cells

T cells are central mediators of adaptive immunity and contribute significantly to graft rejection and tolerance following ITx. CD4+ Th1 cells promote the activation of M1 macrophages via IFN-γ and upregulate iNOS, resulting in apoptosis of intestinal epithelial cells[91,92]. Th17 cells impair mucosal barrier integrity by secreting IL-17A, which downregulates tight junction proteins such as occludin, leading to cryptitis and villous atrophy[93]. Kroemer et al[94] identified elevated levels of TNF-α and IL-17 in CD4+ T cells within rejecting grafts unresponsive to ATG, and demonstrated that anti-TNF-α agents (e.g., infliximab) could reverse these rejections, highlighting the Th17/TNF-α axis as a potential therapeutic target.

CD8+ cytotoxic T cells initiate direct killing of donor epithelial cells via the perforin-granzyme pathway, manifesting histologically as crypt cell apoptosis and epithelial desquamation[95]. Tissue-resident memory T cells (Trm) constitute a long-lived subset residing in the intestinal mucosa and can persist in the graft microenvironment for years. Donor-derived Trm have been shown to survive in recipient intestines for up to 5 years and exhibit dual immunologic functions. While they may participate in early rejection episodes, their long-term persistence also supports the induction of graft tolerance. Bartolomé-Casado et al[96] reported high β2-integrin expression in donor Trm by single-cell RNA sequencing, a potential marker for predicting rejection. Fitzpatrick et al[97] further demonstrated that β2-integrin+ CD8+ Trm cells were enriched in the rejection microenvironment and associated with immune cell infiltration and proinflammatory signaling. Weiner et al[98] revealed that Trm cells harboring host-reactive T cell clones could exacerbate GVHD-like responses, particularly when enriched in the graft. As such, therapeutic strategies aimed at modulating Trm may represent a novel frontier in ITx immunotherapy.

In addition to adaptive immune responses, innate immune cells also play a crucial role in graft injury during AR. Neutrophils exacerbate IRI via the release of NETs, which activate plasmacytoid dendritic cells through TLR9 signaling, promoting activation of the IL-23/IL-17 axis and amplifying early inflammatory responses[99,100]. Additionally, donor-derived NK cells may contribute to rejection by targeting graft endothelial cells through MHC-I mismatch recognition via the "missing self" mechanism[101]. Together, these innate and adaptive immune cells form an integrated effector network that drives AR in ITx.

Regulatory immune cells

Conversely, regulatory T cells (Tregs) are instrumental in promoting immune tolerance. Tregs (CD45+/CD25+/FOXP3+) suppress effector T cell activation through anti-inflammatory cytokines such as IL-10 and TGF-β, thereby maintaining mucosal homeostasis and reducing rejection risk[102]. In particular, donor-specific Tregs play a critical role in facilitating long-term tolerance. The Leuven protocol, for instance, has demonstrated enhanced graft survival by inducing Treg expansion under reduced immunosuppressive conditions[103]. Elevated circulating Treg levels correlate with FoxP3 expression and suppression of DC co-stimulation via CTLA-4-mediated downregulation of CD80/CD86, thereby attenuating effector T cell proliferation[104,105]. The role of FOXP3 is further supported by IPEX syndrome, in which its mutation results in profound autoimmune enteropathy, highlighting its essential role in intestinal immune regulation[106]. Tregs also exert suppressive effects through the enzymatic conversion of extracellular ATP to adenosine via CD39 and CD73, with elevated adenosine levels positively associated with prolonged graft survival[107]. Tregs also exert suppressive effects through the enzymatic conversion of extracellular ATP to adenosine via CD39 and CD73, with adenosine levels positively associated with graft survival[108,109]. M2 macrophages, in turn, promote Treg expansion via TGF-β and reinforce immunosuppressive feedback by inducing IL-10 secretion, establishing a self-amplifying tolerance loop[110].

Post-transplant, the interplay between donor and recipient T cells creates a dynamic chimeric microenvironment, and the stability of this chimerism profoundly influences rejection risk. Notably, T cell chimerism exceeding 1% is associated with reduced rejection rates, particularly in multi-organ transplants such as liver-ITx[111]. Early rejection is typically characterized by clonal expansion of recipient-derived T cells, whereas stable graft acceptance correlates with donor T cell predominance. Furthermore, recipients with low DSA titers exhibit higher likelihood of tolerance induction, potentially due to the regulatory effects mediated by Tregs[111].

Understanding the dual roles of immune cells in both immunogenicity and tolerance is crucial for developing precise, personalized immunomodulatory strategies. Future research should leverage single-cell multi-omics technologies to elucidate the complex cellular networks the ITx microenvironment and guide the design of personalized immune therapies.

The immunological correlation between ITx and the gut microbiota

The gut microbiota plays a dynamic, bidirectional role in post-ITx immune homeostasis, making it a critical determinant of transplantation outcomes. On one hand, microbial translocation can trigger systemic inflammation and exacerbate graft rejection. Following ITx, the intestinal barrier is compromised by IRI, surgical disruption of epithelial integrity, and impaired epithelial renewal resulting from immunosuppressive therapy. These factors facilitate bacterial and metabolite translocation-such as lipopolysaccharide (LPS), peptidoglycan, and lipoteichoic acid-into mesenteric lymph nodes and systemic circulation[36,112-114]. These PAMPs are recognized by pattern recognition receptors (PRRs) on intestinal epithelial and innate immune cells, including TLRs and NOD-like receptors (NLRs)[115-117]. For instance, TLR1, TLR2, TLR4, TLR6, and TLR10 specifically detect microbial ligands such as LPS and lipoproteins. These receptors subsequently activate canonical proinflammatory signaling cascades-most notably the TLR4/NF-κB pathway-which culminate in the release of tumor necrosis TNF-α, IL-6, and IL-1β, thereby amplifying both mucosal and systemic inflammation[118-120].

Additionally, specific bacterial taxa-including segmented filamentous bacteria, Salmonella, and Proteus mirabilis-can drive Th1/Th17-polarized adaptive responses via TLR engagement and antigen presentation pathways, amplifying effector T cell-mediated rejection[121,122]. Post-transplant dysbiosis, particularly the overgrowth of Enterobacteriaceae, has been significantly correlated with a high incidence of acute cellular rejection[123]. Moreover, the translocation of opportunistic pathogens such as Enterococcus faecalis can aggravate graft injury and increase the risk of sepsis[124].

On the other hand, accumulating evidence indicates that certain commensal bacteria may promote graft tolerance by modulating host immunity[125]. For example, Clostridium spp. ferment dietary fiber into SCFAs, such as butyrate and propionate, which activate G protein-coupled receptors (e.g., GPR43) and inhibit histone deacetylases, ultimately enhancing peripheral Treg differentiation and suppressing of Th1/Th17 responses[126-128]. Meanwhile, Lactobacillus species enhance IL-22 secretion via aryl hydrocarbon receptor signaling and contribute to epithelial regeneration[129,130]. In experimental models, pretreatment with Clostridium butyricum has been shown to significantly prolong graft survival and reduce rejection rates in murine ITx models[131]. These findings suggest that select commensal strains may hold translational value in ITx by exerting unique immunomodulatory effects, as further summarized in Table 2.

Table 2 The immune characteristics of intestinal specific microbiota and their correlation with small intestine transplantation.

Bacterial genus	Specific species	Immune mechanism	Receptor/pathway	Association with intestinal transplantation	Potential applications/risks	Ref.
Bifidobacterium	B. longum	Promotes mucosal immune maturation, enhances epithelial integrity	TLR2/4	Loss post-transplant linked to GVHD predisposition	Probiotic supplementation may mitigate GVHD risk	[234,235]
	B. infantis	Balances Th1/Th2 response, modulates cytokine milieu	GPR43 (SCFA-mediated)	Suppresses systemic inflammation, fosters tolerance	May enhance immunosuppressive efficacy	[236]
Lactobacillus	L. plantarum	Downregulates pro-inflammatory cytokines (IL-6, IL-8)	TLR2	Limits endotoxemia and systemic inflammation	Helps prevent post-transplant sepsis	[237]
	L. reuteri	Promotes IL-10 production and Treg induction	AhR	Enhances barrier repair, reduces mucosal inflammation	Therapeutic for immune enteropathies	[238]
Bacteroides	B. fragilis	Induces Treg via PSA antigen	TLR2	Shown to promote tolerance in transplant models	Potential microbial immunotherapy target	[239]
	B. thetaiotaomicron	Promotes IgA secretion, modulates dendritic cell response	TLR4	Supports mucosal immunity, limits pathogen overgrowth	May prevent Enterobacteriaceae dominance	[240,241]
Escherichia coli	Commensal E. coli	Activates basal innate immunity	TLR5/MyD88	Maintains immune tone, but overgrowth risks rejection	Surveillance for strain virulence needed	[242]
	Pathogenic E. coli (e.g., EHEC)	Secretes shiga-like toxins, induces strong cytokine storm	GB3 receptor	Triggers mucosal necrosis, worsens ischemia-reperfusion injury	Requires antibiotic prophylaxis	[243]
Clostridium	C. perfringens	Produces α-toxin, damages tight junctions	-	Can cause graft necrosis, sepsis	Early diagnosis critical	[244]
	C. difficile	Induces pseudomembranous colitis via toxin A/B		Common post-transplant pathogen	Fecal microbiota transplantation emerging as a salvage therapy	[245]
Fusobacterium	F. nucleatum	Activates NF-κB, enhances IL-6/IL-8 release	TLR2/4	Linked to chronic inflammation and neoplasia	Considered a pro-inflammatory marker	[246,247]
Enterococcus	E. faecalis	Inhibits inflammasome, alters antimicrobial peptide balance		Contributes to barrier disruption and bacteremia	Targeted decolonization recommended	[248]
Prevotella	P. copri	Modulates Th17 response		Implicated in dysregulated immunity post- intestinal transplantation	Microbiome-guided regulation needed	[249]
Akkermansia	A. muciniphila	Enhances mucus production, improves tight junctions	GPR43	Shown to improve gut permeability and glucose homeostasis	Probiotic candidate under investigation	[250]
Roseburia	Roseburia spp.	Butyrate production, anti-inflammatory effect	FFAR3 (GPR41)	Maintains Treg differentiation and epithelial repair	Supports mucosal tolerance post-transplant	[251]
Sutterella	Sutterella spp.	Alters IgA response and epithelial interaction		Poorly understood but potentially modulates rejection	Requires further research	[252]

B. longum: Bifidobacterium longum; B. infantis: Bifidobacterium infantis; L. plantarum: Lactiplantibacillus plantarum; L. reuteri: Limosilactobacillus reuteri; B. fragilis: Bacteroides fragilis; B. thetaiotaomicron: Bacteroides thetaiotaomicron; E. coli: Escherichia coli; C. perfringens: Clostridium perfringens; C. difficile: Clostridioides difficile; F. nucleatum: Fusobacterium nucleatum; E. faecalis: Enterococcus faecalis; P. copri: Prevotella copri; A. muciniphila: Akkermansia muciniphila; EHEC: Enterohemorrhagic Escherichia coli; GVHD: Graft-vs-host disease; IL-1: Interleukin-1; SCFA: Short-chain fatty acid; Treg: Regulatory T cell; PSA: Prostate-specific antigen; TLR: Toll-like receptor; NF-κB: Nuclear factor kappa-B.

Broad-spectrum antibiotics such as vancomycin and meropenem, though routinely used to prevent post-ITx infection, profoundly disrupt gut microbial composition and may impair immune homeostasis[132]. Retrospective studies have demonstrated that long-term use of broad-spectrum antibiotics reduces microbial diversity-particularly decreasing Clostridium spp. - and correlates with increased AR incidence[133,134]. Therefore, preoperative microbiome profiling to guide narrow-spectrum antibiotic selection, along with postoperative probiotic supplementation, may help restore microbial balance and improve graft outcomes.

In summary, the bidirectional regulation of immune responses by the intestinal microbiota-via both proinflammatory and tolerogenic pathways-suggests that clinical management of ITx should strike a balance between infection control and microbial homeostasis. A future-oriented therapeutic paradigm could integrate microbiome sequencing, tailored antibiotic regimens, and targeted microbial therapies, ultimately shifting from immunosuppression alone to precision “immune-microbiota co-regulation”.

The immune rejection challenge of ITx

The immunologic complexity of ITx stems from the small intestine’s unique anatomical structure and highly immunoactive environment. As the largest lymphoid organ in the body, the small intestine harbors a dense network of gut-associated lymphoid tissue and resident immune cells, contributing to a heightened risk of rejection compared to other solid organs[135]. This unique immune milieu endows ITx with three hallmark immunological features: (1) Bidirectional immune reactivity, characterized by both HVGR and GVHD. A high proportion of naïve donor T cells within passenger lymphocyte populations serves as the principal effector population for GVHD, particularly in multivisceral transplants[136]; (2) Microbiota-immune interactions, whereby disruption of intestinal flora post-transplant can influence immune activation. For example, the expansion of Enterococcus is closely associated with increased rejection risk and can exacerbate mucosal damage through innate immune signaling pathways, as previously described[137]; and (3) Microbiota-immune interactions, whereby disruption of intestinal flora post-transplant can influence immune activation. For example, the expansion of Enterococcus is closely associated with increased rejection risk and can exacerbate mucosal damage through innate immune signaling pathways, as previously described[138].

Immunological mechanisms of AR in ITx: AR after ITx is driven by a convergence of multiple allorecognition pathways, forming an intricate and partially redundant immunological network (Figure 2). The direct pathway dominates the early phase of AR, where donor antigen-presenting cells (APCs) directly activate recipient CD8+ and CD4+ T cells via donor MHC molecules. This leads to the generation of cytotoxic T lymphocytes (CTLs) and the release of pro-inflammatory cytokines, exacerbating epithelial injury and crypt apoptosis[138,139]. In parallel, the indirect pathway, where recipient APCs process and present donor antigens, promotes CD4+ T cell activation and facilitates the development of DSAs via B cell differentiation-central to CR and AMR[140]. The semi-direct pathway is mediated by donor-derived vesicles or apoptotic fragments containing intact MHC-peptide complexes, which are transferred to recipient APCs. This dual-mode recognition simultaneously activates T cells and promotes humoral sensitization[141-143]. A fourth route, the inverted direct pathway, involves donor CD4+ T cells directly stimulating recipient B cells, accelerating the formation of DSAs and potentially contributing to early-stage rejection not effectively controlled by T cell-targeted immunosuppression[144]. DSAs are identified in approximately 30% of ITx rejection episodes and mediate injury through complement-dependent cytotoxicity (CDC) and ADCC[74]. In CDC, DSAs initiate the classical complement cascade via C1q binding, forming membrane attack complexes that disrupt graft endothelial integrity. In ADCC, DSAs engage Fcγ receptors on NK cells or macrophages, triggering targeted destruction of the graft epithelium[145,146]. Importantly, preformed high-titer DSAs are predictive of AR within the first year post-transplant[147], and their persistent presence can provoke thrombotic microangiopathy through sustained endothelial activation and microvascular injury[148]. Collectively, these findings support the model where the direct pathway primarily drives early AR, while the indirect and semi-direct pathways underpin chronic and late-stage immune responses[149]. Nevertheless, the overlap and synergy among these pathways challenge the efficacy of conventional immunosuppressive regimens.

Figure 2 Schematic representation of the multiple pathway activation mechanisms of the anti-graft response in the intestinal transplant host. Pathway 1: Direct pathway [donor antigen-presenting cell (APC) presenting donor MHC]; Pathway 2: Indirect pathway (recipient APC presenting donor MHC acquired by endocitic pathway); Pathway 3: Semi-direct pathway (recipient APC presenting donor MHC acquired by membrane vesicles); Pathway 4: Inverted direct pathway (donor T cells is activated by recipient MHC presented by recipient B cells). Organized lymphoid tissue in the graft significantly enhances the alloimmune response. It should be noted that pathways 1-3 can occur both in the absorptive mucosa and in the organized lymphoid tissue. All four pathways are activated in the early post-transplantation period, whereas only pathways 2 and 3 persist in long-surviving grafts. APC: Antigen-presenting cell.

In addition to recognition pathways, the spatiotemporal orchestration of effector immune cells critically dictates rejection outcomes. CD4+ and CD8+ T cells are the dominant cellular mediators in AR, with CD4+ T cells differentiating into Th1 and Th17 phenotypes. Th1 cells secrete IFN-γ and TNF-α to activate macrophages and amplify CTL responses, while Th17 cells drive neutrophil recruitment and local tissue inflammation through IL-17A and IL-21[150-153]. An increased frequency of Th17 cells within the lamina propria correlates with histopathologic severity in AR, suggesting a pathogenic role for the IL-23/IL-17 axis in mucosal injury[154].

Concurrently, innate immune responses substantially contribute to the onset of AR. After transplantation, the exposure of intestinal tissue to microbial products rapidly activates PRRs such as TLRs and NLRs on innate immune cells[155]. Variants in the TLR4 gene have been linked to altered susceptibility to rejection, likely due to dysregulated responses to LPS[156]. In clinical observations, a high density of MPO+ neutrophils within graft biopsies is significantly associated with severe AR episodes and poor outcomes[157].

Together, these findings underscore the multifactorial nature of immune rejection in ITx, highlighting both adaptive and innate contributors, as well as complex crosstalk between cellular and humoral effectors. Future strategies must shift toward precision immunotherapy, integrating real-time immune monitoring and pathway-specific interventions to achieve durable graft survival.

Immunological mechanisms of CR in ITx: CR following ITx is driven by a multifaceted immunological cascade, characterized by persistent low-grade inflammation and progressive tissue remodeling. At the cellular level, both CD4+ and CD8+ T lymphocytes recognize donor MHC antigens via direct presentation or donor peptide-MHC complexes processed through the indirect pathway by recipient APCs[158]. The resulting T cell activation leads to elevated secretion of proinflammatory cytokines such as IFN-γ and IL-17, establishing a Th1/Th17-skewed microenvironment that promotes endothelial apoptosis and crypt epithelial injury[159,160]. Effector memory T cells, notable for their long-term persistence post-transplant and reduced susceptibility to immunosuppressive agents, may underlie the insidious progression and treatment resistance observed in CR[161].

On the humoral front, DSAs bind to endothelial antigens via their Fab regions, initiating complement activation (evidenced by C4d deposition) and Fcγ receptor-mediated immune responses through their Fc domains[162,163]. Emerging evidence suggests that the continuous presence of DSAs may stem from long-lived plasma cells that evade immunosuppression, resulting in ongoing B-cell-mediated injury and immune amplification[164].

Innate immunity plays an increasingly recognized and synergistic role in CR. Macrophage polarization exhibits stage-dependent functional divergence: In early rejection, IFN-γ-driven M1 macrophages secrete high levels of TNF-α and IL-6, fueling local inflammation; whereas in late-stage rejection, M2 macrophages promote fibrogenesis and tissue remodeling via secretion of TGF-β[165,166]. NK cells contribute to vascular injury through ADCC, while neutrophils promote microvascular thrombosis by releasing NETs.

The pathological hallmark of CR is chronic inflammation-induced fibrosis, orchestrated by a dense cytokine signaling network. TGF-β activates myofibroblasts and promotes their transdifferentiation via the canonical Smad3 pathway, while platelet-derived growth factor enhances their proliferation through the PI3K/Akt signaling axis[167,168]. In advanced stages, Th2-biased immunity becomes dominant. IL-13, via STAT6 signaling, upregulates fibrogenic gene expression (e.g., COL1A1, TIMP1), exacerbating matrix accumulation and structural distortion of the graft[169,170]. Notably, IL-13 knockout significantly attenuates fibrosis in animal models, supporting its pathogenic role[171]. A key pathological amplifier in CR is vascular injury-induced ischemia. Histopathological studies have shown that chronically rejected intestinal grafts exhibit typical intimal hyperplasia and arterial luminal narrowing resembling atherosclerosis[172-174]. The consequent hypoperfusion activates hypoxia-inducible factor-1α, promoting pro-fibrotic signaling and forming a self-perpetuating cycle of ischemia and fibrosis[175].

Importantly, immunosuppressive escape presents a major clinical barrier in CR. The lymphoid-rich architecture of the small intestine permits persistent donor leukocyte chimerism, predisposing to GVHD and sustained alloimmune activation[176]. Concurrently, microbial dysbiosis-especially loss of commensals and overgrowth of opportunistic pathogens-can activate TLR/NLR pathways and disrupt immune homeostasis, undermining the efficacy of immunosuppression[177]. Beyond immunological mechanisms, several non-immunologic factors also contribute to CR. IRI, a key upstream insult, triggers DAMPs such as high-mobility group box protein 1 (HMGB1), which activate NF-κB via TLR4 and RAGE, promoting cytokine storm and chronic inflammation[178,179]. Surgical factors, including lymphatic disruption and autonomic denervation, may also alter mucosal immune dynamics, aggravating chronic allograft dysfunction[180].

In summary, CR in ITx arises from a complex interplay network of adaptive and innate immune interactions, stromal-vascular remodeling, and extrinsic surgical insults. This underscores the urgency to develop multi-targeted interventions that transcend traditional immunosuppression, incorporating anti-fibrotic, anti-hypoxia, and microbiome-based strategies.

INNOVATIONS IN IMMUNOTHERAPEUTIC STRATEGIES

Induction and maintenance immunosuppression

To mitigate immune rejection after ITx, current clinical strategies adopt a phase-based immunosuppressive approach encompassing induction and maintenance therapy, tailored to the recipient’s immune risk profile. For induction therapy, commonly used agents include ATG, basiliximab (anti-IL-2R), and alemtuzumab (anti-CD52). Among these, ATG remains the mainstay, particularly in high-risk recipients due to its potent T-cell depletion capacity[181]. Basiliximab, with a more favorable safety profile and reduced infection risk, is often preferred in non-sensitized patients and allows for corticosteroid minimization[103,182]. Alemtuzumab may offer protection against GVHD, but its association with PTLD and infections restricts its broader application[183,184].

The cornerstone of maintenance therapy continues to be tacrolimus, often in combination with corticosteroids. Mycophenolate mofetil is frequently incorporated to reduce calcineurin inhibitor-induced nephrotoxicity[185]. Sirolimus, via mTOR inhibition, has shown potential in late-phase immunosuppression and may provide additional protection against CR and GVHD[186]. In cases of AR, high-dose corticosteroids remain first-line; for steroid-refractory rejection, ATG or targeted biologics such as vedolizumab (anti-α4β7 integrin) and infliximab (anti-TNF-α) are employed. Notably, infliximab has demonstrated efficacy in suppressing Th17-mediated inflammation, offering a viable option for steroid-resistant AR[94].

Emerging biologics and cell therapies

Nevertheless, immunosuppressive toxicity presents a major clinical challenge. Long-term tacrolimus use results in renal impairment in up to 50% of recipients, typically reflected by a > 30% decline in glomerular filtration rate[187]. Treatment resistance is frequently driven by long-lived TEM and plasma cells evading immunosuppression, perpetuating DSA production and AMR[188]. Genetic polymorphisms in CYP3A5 contribute to substantial variability in tacrolimus pharmacokinetics, complicating drug level optimization[189]. Furthermore, persistent activation of TGF-β/Smad signaling promotes irreversible fibrotic remodeling, underlining the elevated risk of late graft loss[190]. A persistent clinical dilemma remains: Intensifying immunosuppression curbs rejection but increases viral reactivation (e.g., cytomegalovirus, Epstein-Barr virus), while de-escalation may trigger recurrent rejection episodes, posing a therapeutic paradox for clinicians[191].

Recent advances in targeted immunotherapies offer new hope. In B-cell-directed strategies, rituximab (anti-CD20) reduces DSA levels and improves graft survival[192]. Bortezomib, a proteasome inhibitor targeting plasma cells, has demonstrated efficacy in AMR, though it carries the caveat of heightened infection risk, necessitating cautious use in the ITx setting[193,194]. JAK inhibitors such as ruxolitinib have shown benefit in steroid-refractory GVHD by dampening JAK-STAT-mediated proinflammatory cytokine signaling[16,17]. For modulation of innate immunity, TAK-242-a TLR4 inhibitor-has been shown to ameliorate IRI by blocking HMGB1-TLR4 signaling[195]. Targeting macrophage polarization is another promising avenue: Colony-stimulating factor-1 receptor inhibitors selectively deplete profibrotic M2 macrophages and attenuate intestinal fibrosis in experimental models[196,197].

Microbiome-based and technological interventions

Microbiome-based interventions are rapidly gaining traction. FMT has been shown to restore microbial diversity, elevate IL-25 Levels, and promote mucosal repair, offering intestinal protection[18]. Specific probiotic strains can augment Tregs via SCFA production, reducing AR incidence[198]. Moreover, FMT and probiotics have demonstrated therapeutic potential in GVHD, expanding their role beyond infection prevention.

In humoral rejection, the complement inhibitor eculizumab targets C5 convertase, effectively reducing C4d deposition in graft endothelium, representing a novel therapy for AMR[199]. Concurrently, advances in tissue engineering provide innovative approaches for CR. Decellularized intestinal scaffolds, preserving native extracellular matrix and vascular architecture, when seeded with mesenchymal stem cells, promote mucosal regeneration and attenuate fibrosis in preclinical models[200,201].

Emerging diagnostics enhance early rejection surveillance. Exosomal miR-155-5p outperforms traditional histology in detecting AR and holds promise as a noninvasive biomarker[202]. Luminex-based multiplex assays enable precise DSA quantification, with studies linking mean fluorescence intensity > 10000 to a fourfold increase in CR risk[203]. Artificial intelligence is reshaping transplant monitoring-deep learning models integrating histological and transcriptomic data have achieved 92% predictive accuracy for rejection events, offering robust decision-making support[204].

Future directions will likely emphasize multi-target synergistic immunoregulation. Experimental evidence suggests that donor-derived hematopoietic chimerism combined with targeted cellular therapy enables successful weaning of immunosuppression without rejection, representing a path toward “immune independence”[205,206]. A combinatorial strategy involving JAK inhibitors, anti-fibrotic agents, and microbiome modulation may allow synchronized regulation of adaptive immunity, fibrosis, and the gut-immune axis. Additionally, gene-editing technologies such as CRISPR-Cas9 to delete MHC class I genes in donor organs have prolonged graft survival in models, indicating transformative potential[207]. Finally, neuroimmune modulation is an emerging frontier. A clinical trial investigating vagus nerve stimulation aims to suppress proinflammatory cytokines via α7 nicotinic acetylcholine receptor activation, potentially unlocking a novel anti-rejection paradigm[208]. Collectively, these advances signify a paradigm shift toward precision immunotherapy and immune tolerance in ITx.

CHALLENGES AND FUTURE RESEARCH

ITx has evolved into a critical therapeutic strategy for patients with irreversible intestinal failure, especially those unresponsive to PN or facing life-threatening complications. Over the past decades, significant progress has been made in surgical techniques, immunosuppressive strategies, and perioperative management. Advances such as optimized vascular anastomosis, multi-organ transplantation approaches, and NMP have improved short-term outcomes and extended graft viability.

However, ITx continues to face unique and formidable challenges. The small intestine’s exceptional immunogenicity, complex mucosal immune environment, and dense microbial population create a distinctive setting for both HVGR and GVHR. Moreover, chronic immune rejection, the toxicity of long-term immunosuppressive therapy, and persistent postoperative complications remain major barriers to long-term graft survival and patient quality of life.

To overcome these obstacles, future research must embrace a multidisciplinary and translational approach. Key priorities include the development of precision immunosuppressive regimens tailored to individual immune risk, deeper understanding of host-microbiota-immune interactions, and the clinical translation of novel technologies such as gene editing, 3D-bioprinted grafts, immune monitoring using machine learning, and microbiome-targeted therapies. Furthermore, integrating robotic-assisted surgery and tissue engineering into clinical practice may enhance surgical accuracy and minimize complications.

CONCLUSION

In conclusion, the trajectory of ITx is shifting from a last-resort intervention toward a personalized, technology-driven therapy. Through collaborative innovation across surgery, immunology, microbiology, and bioengineering, ITx is expected to become a safer, more effective, and durable solution-ultimately transforming the long-term outlook for patients with complex intestinal failure.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the contributions of the General Surgery Department and all clinicians and researchers involved in this study.