Introduction: 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) has yet to be defined.

Methods: Lamina propria 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, and CD127. Four distinct subsets were further defined as NKp44-ILC1, NKp44-ILC3, NKp44+ILC1 and NKp44+ILC3.

Results: We compared ILC phenotypes via flow cytometry in stable ITx recipients with healthy functioning allografts >6 months after ITx to fresh ITx recipients at day 0 after reperfusion. Surprisingly, 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, NKp44+ILC1) and NKp44-ILC3 in both fresh and stable ITx recipients, indicating a dysbalance between protective and proinflammatory ILC subsets in fresh but not stable recipients. Intracellular cytokine staining confirmed that NKp44+ILC3 produced protective IL-22, while ILC1s and NKp44-ILC3 produced pro-inflammatory IFN-g, TNF-α, and IL-17. Importantly, serial prospective immunomonitoring of fresh ITx recipients revealed that protective NKp44+ILC3 repopulates by 1 month postoperatively, suggesting that protective and proinflammatory ILCs re-equilibrate in ITx patients over time. Critically, the frequencies of repopulating protective NKp44+ILC3 correlated positively with IL-22 dependent antimicrobial peptide (AMP) expression including β-defensins and RegIIIγ, which are important for intestinal barrier protection. In line with this, we further noted that there was a significant increase in the frequency of protective NKp44+ ILC3 in healthy ITx tissue compared to inflamed tissue 1 month after ITx.

Conclusion: Our study indicates that reconstitution of protective ILC3, which is absent immediately post transplant, positively correlates with improved epithelial barrier function through increase of IL-22 dependent AMP expression. In contrast, abundance of proinflammatory ILC1 and ILC3 may be contributing to epithelial barrier breakdown and early clinical complications.