Mucosal Mixed Lymphocyte Reaction Assay Using Intestinal Lymphocytes as a Biomarker for Intestinal Transplant Tolerance Development

Background. Intestinal transplantation (ITx) has the highest rate of rejection among solid organ grafts. We aimed to study the pathophysiology of rejection after ITx but lacked a tool for assessing cellular responses within the graft. Therefore, we developed a novel mixed lymphocyte reaction (MLR) assay to investigate immune responses in the graft. Methods. Intestinal samples were collected, decontaminated, and processed into single-cell suspensions from 9 swine and 2 patients that underwent ITx. Debris was removed using gradient centrifugation. The cells were plated with corresponding stimulator cells and incubated for 6 d before data acquisition and analysis. Results. Tolerant animals showed no anti-donor or anti-recipient responses in their graft mucosa but maintained strong anti–third-party responses, even after weaning immunosuppression. An animal with graft-versus-host disease displayed robust anti-recipient and anti–third-party responses but no anti-donor response. The animals with graft rejection maintained anti-donor responses at all timepoints. Finally, some tolerant animals developed “split tolerance,” with anti-donor responses in the peripheral blood but donor-specific hyporesponsiveness in the mucosal MLR, which regulatory T cells depletion suggested was attributable to local regulatory tolerance. When applied to human sample, this mucosal MLR reliably demonstrated self-tolerance with normal anti–third-party responsiveness. Conclusions. The novel mucosal MLR assay presented herein ± CD25 depletion serves as a useful adjunct for assessing immune responses within the intestinal graft mucosa. This could help elucidate immune responses after ITx in future studies, including our own, and could represent a promising tool for studying ITx tolerance development, guiding immunosuppression strategies, and advancing personalized transplant medicine.