**Background:** Intestinal transplantation (ITx) is a life-saving option for patients with irreversible intestinal failure and complications from total parenteral nutrition. Since its clinical establishment in the late 1990s, ITx has been recognized as the most immunogenic solid organ transplant, requiring more aggressive immunosuppression than other organ transplants due to the graft's constant exposure to external antigens, a diverse microbiota, and a large load of tissue-resident immune cells. Acute cellular rejection (ACR) occurs more frequently than in any other organ transplant and can rapidly lead to mucosal barrier loss, sepsis, and death. Despite this, no noninvasive biomarker has demonstrated adequate sensitivity and specificity to replace endoscopic biopsy for ACR diagnosis. This review integrates current knowledge on ITx immunobiology with mechanistic aspects of graft rejection and evaluates emerging candidate biomarkers.
**Methods:** This is a narrative review synthesizing experimental and clinical literature on ITx immunobiology and biomarkers. The authors discuss foundational murine studies from the late 1990s/early 2000s by Newell and coworkers, recent work from Megan Sykes' lab on two-way alloreactivity, and clinical studies on donor-specific antibodies (DSA), fecal calprotectin, plasma citrulline, and omics-based approaches. Key experimental models include murine skin and kidney transplant models for studying resident memory T cells (Trm), rat multivisceral transplant models, and human biopsy and biofluid analyses from centers including Columbia University and Swedish institutions.
**Key Results:** The review identifies four allorecognition pathways in ITx: (1) direct pathway—donor APC presentation of alloantigens to recipient CD4+ and CD8+ T cells, mainly responsible for early ACR; (2) indirect pathway—recipient APC presentation of donor peptides, operative indefinitely and linked to DSA generation; (3) semidirect pathway—transfer of donor MHC molecules via extracellular vesicles to recipient APCs; and (4) inverted direct pathway—donor CD4+ T cells activating recipient B cells, contributing to early de novo DSA formation. The two-way alloreactivity model shows concurrent expansion of graft-versus-host (GvH) and host-versus-graft (HvG) T cell clones under immunosuppression, with overexpansion leading to clinical rejection or GvHD. Trm cells, particularly CD8+ and Th17-like CD4+ Trm, are identified as central players in chronic rejection, with alloreactive Trm from skin grafts capable of rejecting heterotopic heart grafts in murine models. In clinical ITx, Th17 cells with a memory phenotype were described in thymoglobulin-resistant rejection. For biomarkers: plasma citrulline shows sensitivity >80% but specificity of only 58% for ACR, cannot distinguish rejection from infectious enteritis, and is confounded by renal function and graft size. Fecal calprotectin at a cutoff of 92 mg/L shows 83% sensitivity and 77% specificity for ACR but is elevated in both rejection and non-specific enteritis. Granzyme B and perforin mRNA in PBMCs show 80% and 70% sensitivity and 87% and 87% specificity, respectively, but are also elevated in post-transplant lymphoproliferative disease and viral enteritis. Serum ST2 at a cutoff of 3150 pg/mL shows 62% sensitivity and 72.2% specificity for rejection. Metabolomic analysis of stomal effluent identified 477 of 2541 detected metabolites with significant fold change between rejection and nonrejection, with leukotriene E4, D-pantethine, pyridoxal-5-phosphate, taurocholate, and riboflavin showing the most significant changes. Proteomics in murine rejecting allografts identified 86 differentially expressed proteins (37 upregulated including S100-A8 and IDO-1; 49 downregulated including chromogranins and ornithine aminotransferase). Microbiome analysis during ACR shows a reduction in Firmicutes (from 81% to 29%) and expansion of Proteobacteria (from 16% to 61%) at the phylum level.
**Clinical Implications:** The review concludes that no single noninvasive biomarker currently stands alone as a diagnostic assay for intestinal ACR. Plasma citrulline and fecal calprotectin are in clinical use but only as screening or exclusionary tools, with citrulline reflecting the extent of mucosal injury rather than serving as a specific diagnostic marker. The identification of Trm as central to chronic rejection has important implications, as immunosuppressive drugs have lower activity on memory cells compared to naïve cells. The two-way alloreactivity model suggests that monitoring chimerism dynamics could allow adjustment of immunosuppression or development of tolerogenic strategies. The association between de novo DSA and poorer long-term outcomes supports DSA monitoring as an indicator of ongoing immunologic activity. The authors advocate for future multicenter studies using high-throughput omics technologies to develop composite biomarker panels, similar to the metabolite–mRNA signatures being developed in kidney transplantation, and emphasize the need to explore circulating cell-free donor DNA and microbiota alterations as potential early biomarkers.