Introduction: Severe allograft rejection is one of the strongest prognostic factors leading to eventual graft loss and mortality in intestinal transplantation (ITx), specifically if the rejection is refractory to traditional therapeutic maneuvers such as T cell depletion with thymoglobulin(thymo). A characterization of the alloreactive T cell phenotype in severe rejection of both thymo treatment responsive and non-responsive patients has been lacking and is key for unlocking a precision medicine approach.

Methods: A cohort of 57 ITx patients with severe cellular rejection both before, during, and after treatment with thymo, alongside 128 uncomplicated controls, was selected from our IRB-approved Immunomonitoring and Tissue Bank Study. A polychromatic flow cytometry (PFC) panel with and without PMA/Ionomycin re-stimulation and culture was used to analyze peripheral blood and intestinal allograft samples to characterize surface receptor phenotype and cytokine production.

Results: Immunomonitoring of blood via PFC revealed that peripheral CD3 T cells uniformly deplete in both responders and non-responders (p=.47), confirming that monitoring CD3 T cells in peripheral blood fails to correlate with treatment responsiveness. However, CD3 IHC analysis of rejecting allografts confirmed that the degree of T cell depletion in the allograft correlates with clinical responsiveness. Graft immunomonitoring via PFC showed CD4+ effector memory, terminally differentiated effector memory, and pro-inflammatory CD4+ Th17 cells persist both before and during treatment with thymo. PFC cytokine profile evaluation using ex vivore-stimulated T cells showed a significant increase in IL-17 production (p<.05) from the Th17 cell population in rejection versus controls, which was also corroborated by rtPCR-arrays demonstrating a striking increase in the Th17-related transcriptome signature in rejection vs controls. Moreover, the Th17 cells of these patients co-expressed high levels of TNF-α, reminiscent of immunological features of inflammatory bowel disease (IBD). Indeed, when 10 non-responder patients were treated with the anti-TNF-α IBD medication infliximab, 9 experienced histologic and clinical recovery from rejection.

Conclusions: Severe ITx rejection is characterized by a Th17-mediated alloimmune response. Given the immunological similarities to IBD, we postulate that severe ITx rejection can be considered a third IBD, which has important clinical treatment implications.