*Purpose: Innate lymphoid cells (ILCs) play fundamental roles in mucosal barrier functionality and tissue homeostasis. We hypothesized that protective IL-22-producing NKp44⁺ILC3 subsets are reduced under inflammatory conditions in intestinal transplantation (ITx) thus affecting downstream genes associated with intestinal homeostasis and ILC effector’s regulation.

*Methods: To test our hypothesis, polychromatic flow cytometry (PFC) and RNA-seq experiments were performed on serial ITx biopsy samples from healthy, stable ITx patients. For PFC, 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. For RNA-seq, total RNA was isolated from frozen tissue and libraries were sequenced on the NextSeq 550 System.

*Results: ILC phenotype analysis via PFC revealed that NKp44⁺ILC3s were significantly diminished under inflammatory conditions at the day-of ITx in allografts after reperfusion (d0) compared to stable recipients 6 months out. Critically, the NKp44⁺ ILC3 subsets were also decreased in postoperatively inflamed allografts compared to healthy controls. To investigate if reduced NKp44⁺ILC3s could affect genes associated with intestinal homeostasis and integrity, we performed RNA-seq analysis using serial ITx biopsies and compared the transcripts in d0 allografts vs. healthy 2-4 weeks’ and healthy> 6 months’ allografts. From RNA-seq analysis, we identified 5,085 differentially expressed genes (DEGs) (false discovery rate, FDR<0.05) at d0 vs. >6 months and 2,895 DEGs at d0 vs. 2-4 weeks, respectively. Importantly, we found a markedly lower expression of genes involved in NKp44+ILC3-related intestinal barrier viability and homeostasis pathways, including IL-22, IL-23, NOD2, IL1B, S100A8, and S100A9 in inflamed d0 allografts in comparison to healthy allografts.

*Conclusions: Protective NK44⁺ILC3s were diminished in d0 allografts at the time of ITx compared to healthy allografts >6 months post-ITx. RNA-seq analysis identified altered genes associated with mucosal defense and intestinal homeostasis in day 0 vs. healthy ITx allografts. Our findings may provide insights into clinical implications for the early prediction and clinical management of intestinal transplantation.

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