Split Tolerance in Intestinal Transplantation: Promising Translational Evidence With Relevance for Daily Clinical Practice

Over the years, the achievement of graft tolerance has been the “holy grail” for clinical transplantation, particularly in 1 of the most challenging organs to be transplanted, the intestine.1 Although different experimental approaches and clinical reported experiences have resulted in the accumulation of knowledge about possible mechanisms and strategies that favor the establishment of tolerance, autonomous allograft tolerance has not been able to be achieved yet. The pursuit of tolerance in intestinal transplantation has been both fascinating and challenging because of several unique features of this organ; the intestinal graft harbors a significant, compartmentalized lymphocyte load that is introduced into the recipient, and donor lymphoid cells begin circulating in the recipient immediately after reperfusion. Simultaneously, recipient lymphocytes start populating and interacting with the donor cell population in different compartments, forming a true chimera,2,3 capable of preserving structure and function despite immune cell interactions and immunosuppressive treatment.4 These interactions often lead to rejection in most patients, graft-versus-host disease (GVHD) in some, and, in rare instances, successful tolerance.5 Those few cases have been reported as operational tolerance after intestinal transplantation.6 The development of novel diagnostic/monitoring technologies would allow a better understanding of the donor-to-graft and graft-to-donor responses and potential approaches to induce intestinal transplant tolerance.4–6 Achieving genuine immune tolerance—defined as the state in which the immune system does not respond or fails to react to a foreign or self-antigen—after intestinal transplantation has proven extraordinarily difficult. Beyond the multiple alloreactive pathways mentioned earlier, another major constrains in achieving graft tolerance has been the lack of appropriate tools to functionally evaluate tolerance and provide feedback for tolerogenic strategies in patient management.6,7 The recent work of Gunes et al describes a novel mixed lymphocyte reaction (MLR) assay8 that both at the bench and in clinical practice can be used to evaluate the existence of split tolerance. This novel tool could be standardized to diagnose, monitor, and eventually intervene in transplanted intestines using its readout to fine-tune therapy management, maximizing tolerogenic circuits. This finding may become a cornerstone because it facilitates the evaluation of the immune response in the graft and deepens our understanding of the pathways leading to either rejection or tolerance. The published findings were made possible by establishing a successful decontamination, cell isolation, and debris-removal protocol to obtain lymphocytes from the intestinal graft mucosa for evaluating alloreactivity using a classical MLR assay. Although the assay is conceptually simple, there were specific technical challenges that the authors wisely overcame, enabling the development of a robust and rigorous procedure. Given that setting up an MLR reaction using graft-derived lymphocytes challenged with donor cells is the assay’s central component, intestinal or multivisceral grafts are ideal for this type of assay. This is because of the relatively straightforward accessibility to the graft in these transplants and the substantial lymphocyte load found within the graft. One major challenge in this procedure is dealing with cellular and tissue debris after tissue digestion, as obtaining a clean cell suspension is essential for subsequent assay readout. Gunes et al8 optimized the technique in a pig intestinal transplant model by using tissue layer separation, sequential digestion of the mucosal layer and a combination of collagenase and DNAse to minimize cell clump formation. Furthermore, after testing various options, they used a commercial buffer for removing cell and tissue debris, significantly improving subsequent flow cytometry analyses. After optimizing cell separation, the authors demonstrated that the MLR can be performed under various conditions, each providing valuable information. They showed that they could recover sufficient cells to assess antidonor, anti-self, or anti-third-party reactivity. Furthermore, by implementing anti-CD25 depletion of mucosal cells, the authors were able to evaluate the impact of CD25+ regulatory T cells on the outcome of the assay. The proof of concept was confirmed by analyzing graft lymphocyte reactivity in a series of transplanted pigs that developed rejection, GVHD, or no rejection. The results showed that significant antidonor reactivity is predominant in cases of rejection, whereas anti-recipient activity was dominant in GVHD. Most notably, by using a CD25 depletion assay, the authors demonstrated “split tolerance.” Specifically, they detected significant antidonor reactivity in MLR assays performed with peripheral cells but observed no reactivity when using graft mucosal lymphocytes—an absence that disappeared after CD25 depletion. These findings indicate an active tolerance process in the graft sustained by CD25+ regulatory T cells in the mucosa, whereas this population is lacking or insufficient in the periphery to prevent a positive MLR response. Furthermore, the authors showed that this technique can be applied to clinical samples obtained during ostomy closure procedures. In line with this, a growing body of evidence highlights the central role of CD25+ regulatory T cells in establishing and maintaining operational tolerance after intestinal transplantation. Several reports of operationally tolerant recipients describe a robust intragraft regulatory T-cell population, suggesting their pivotal contribution to stable graft acceptance. Although donor-specific regulatory T cells in these patients have not been directly confirmed, their presence in tolerant grafts implies that tolerance may be driven by these cells. One proposed mechanism involves regulatory T cell–mediated suppression of cytotoxic memory T-cell activation. Regulatory T cells also appear to disrupt antigen presentation by directly interacting with antigen-presenting cells, thereby further reducing alloreactivity.6 Consequently, recognizing a mucosal regulatory T-cell response—independent of the systemic immune responses—opens the door to local immune interventions that not only foster the development of graft tolerance9,10 but also facilitate early GVHD diagnosis, reduce reliance on systemic immunosuppression, and mitigate its associated side effects, such as chronic renal disease, hypertension, and diabetes.