Innate Lymphoid Cells are Dysregulated after Intestinal Transplantation and Play a Critical Role in Regulating Allograft Homeostasis

Abstract

The recent discovery of innate lymphoid cells (ILCs) has revealed their importance in intestinal homeostasis; however their role in intestinal transplantation (ITx) has yet to be defined. We hypothesized that ILC subsets are dysregulated in the lamina propria following ITx thus affecting intestinal homeostasis and intestinal stem cell (ISC) regeneration. Intestinal samples were obtained from patients immediately post ITx and at several time points within the first 6 months as well as from stable patients over 6 months post-op without acute complications. Lamina propria cells were isolated and immunophenotyped. Viable lineage negative cells were identified as group 1 and 3 ILCs by expression of CD56, NKp44, CD117, and CD127. Graft infiltrating lymphocytes were flow sorted and rtPCR was performed to investigate effector cytokine and transcription factor profiles. First we compared a group of stable patients with samples from fresh ITx recipients taken immediately after transplant (day 0). Critically, we found that the pro-inflammatory IFN-g-producing CD127+ ILC1 subset (p=0.492) was preserved in fresh ITx and comparable to stable patients. Importantly, however, we found that the pro-homeostatic IL-22-producing NCR+ ILC3 population was absent in fresh allografts when compared to stable recipients (p=0.019). We then hypothesized that the observed ILC1-ILC3 dysequilibrium resolves over time. This was tested by prospective immunomonitoring of ITx patients from the day of transplant onwards utilizing flow cytometry, immunohistochemistry, and rtPCR. Intriguingly, we found that the CD127+ ILC1 population overall remained stable while there was a recovery of the total NCR+ population and ILC3 in complication-free ITx patients after 4–8 weeks post ITx (p=0.025). These results demonstrate that the ILC dysbalance seen post-ITx resolves with the recovery of protective ILC3. This has a potentially profound impact on allograft homeostasis post-ITx as we further found that the recovery of NCR+ cells and ILC3 correlated with significantly enhanced expression levels of LGR5, a marker for IL-22-dependent ISC, by postoperative week 4-8 (p=0.003).In summary, this study first demonstrates a dangerous dysbalance between the pro-inflammatory ILC1s and the protective ILC3s within the first few weeks following ITx, when patients experience most complications. This dysregulation resolves over time with the regeneration of NCR+ ILC3s that target ISCs for allograft recovery.