*Purpose: Innate lymphoid cells (ILCs) , the most recently described family of lymphoid cells, play fundamental roles in mucosal barrier immunity, tissue homeostasis, and immune regulation through the activation of host-derived cytokine expression; however their roles in intestinal transplantation (ITx) have yet to be defined.

*Methods: Twenty-six participants who underwent IT x at our Transplant Institute were enrolled in this study. Intestinal biopsies were obtained immediately after ITx at day 0 and followed longitudinally. LP cells were isolated and the following phenotypic definition was used for ILCs: lineage negative, viable lymphocytes expressing CD45 were identified as type 1 and 3 ILCs by expression of CD56, NKp44, CD117 (c-Kit), and CD127 (IL7Rα). Four distinct subsets were further defined as NKp44^–ILC1 (CD56^–CD127⁺CD117^–), NKp44^–ILC3 (CD56^–CD127⁺CD117⁺), NKp44⁺ILC1 (CD56⁺CD127^–CD117^–) and NKp44⁺ILC3 (CD56⁺CD127⁺CD117⁺). Graft infiltrating lymphocytes were flow sorted and rtPCR was performed to investigate effector cytokine and transcription factor profiles.

*Results: To test our hypothesis, we explored ILC phenotypes via flow cytometry in stable ITx recipients with healthy functioning allografts >6 months after ITx in comparison to fresh ITx recipients at day 0 after reperfusion. To our surprise we found that protective NKp44⁺ILC3s (p=0.02) were significantly diminished in fresh allografts compared to NKp44⁺ILC3s in stable recipients 6 months out. In addition, we found comparable numbers of potentially proinflammatory ILC1s (NKp44^–ILC1 (p=0.18), NKp44⁺ILC1 (p= 0.82)) and NKp44^–ILC3 (p=0.88) in fresh and in stable ITx recipients, indicating a dysbalance between protective and proinflammatory ILC subsets in fresh but not stable ITx recipients. By intracellular cytokine staining, we confirmed that NKp44⁺ILC3s produced protective IL-22, while ILC1s and NKp44^–ILC3sproduced pro-inflammatory IFN-γ, TNF-α and IL-17. Importantly, serial, prospective immunomonitoring of fresh ITx recipients revealed that protective NKp44+ILC3s repopulate by 1 month postoperatively, suggesting that protective and proinflammatory ILCs re-balance in ITx patients over time. Critically, the frequencies of repopulating protective NKp44⁺ILC3s correlated positively with IL-22 dependent antimicrobial peptide (AMP) expression including β-defensins and RegIIIγ which are important for intestinal barrier protection, as determined by rtPCR.

*Conclusions: Our study indicated that the reconstitution of protective ILC3 which are absent immediately post transplant positively correlates with improved epithelial barrier function through increase of IL-22 dependent AMP expression. In contrast, proinflammatory ILC1 and ILC3 subset abundance could be contributing to epithelial barrier breakdown and early clinical complications.

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