Introduction: Regulatory T cells (Tregs) have recently been established as key players in dampening effector T cell (Teff) mediated inflammation, managing self-tolerance, and preventing autoimmunity and transplant rejection. However, the role of Tregs in immune responses in intestinal transplantation (ITx) is unknown, where a full tissue phenotypic and functional characterization of the Treg/Teff axis is lacking. We hypothesized that this axis in ITx rejection is dysbalanced towards unrestricted Teff-mediated inflammation in the allograft.

Methods: We identified a cohort of 17 ITx patients with a history of rejection along with 24 uncomplicated controls from our IRB-approved Immunomonitoring and Tissue Bank Study. A polychromatic flow cytometry (PFC) panel with and without PMA/ionomycin stimulation and culture was used to analyze peripheral blood and intestinal allograft samples to characterize surface receptor phenotype, transcription factor expression, and cytokine production.

Results: PFC of biopsies showed a significant increase in pro-inflammatory IL-17 producing CD4+ Th17 effector cells in rejection patients as compared to controls. To our surprise, there was also an overall increase in the proportion of FoxP3+ Tregs in rejection patients; however, the majority of which were induced (Helios-) with less natural thymic derived Tregs (Helios+), which was not observed in blood. Further Treg subset analysis revealed a significantly higher proportion of pro-inflammatory Th17-like CCR6 expressing and Th1/Th17-like dual CXCR3/CCR6 expressing memory Tregs in rejection patients. Therefore, we hypothesized that given Treg plasticity in a pro-inflammatory environment, these Tregs assume an effector like phenotype in ITx rejection. To test this hypothesis, we performed an ex vivorestimulation assay, which demonstrated that both natural and induced Tregs produce more pro-inflammatory IL-17 and IFN-γ rejection than control patients, further corroborating their pro-inflammatory phenotype and dysbalance in the Treg/Teff axis towards a proinflammatory Th17 phenotype.

Conclusion: Our study characterizes ITx rejection as driven by a severely altered Treg/Teff axis with IL-17 producing pro-inflammatory CCR6+ Th17 effector and potentially pro-inflammatory Treg cells, which may have strong implications on future clinical therapies.