We have shown that differently from native small bowels, NK-like cells (CD45+CD103+CD3-) are the predominant intra-epithelial lymphocytes (IEL) in small bowel grafts (SBG) from 4th to 24th month post-transplant (Tx). To test if these IEL may correspond to the recently described innate lymphoid cells (ILC), IEL from 69 ileal mucosa biopsies (Bx) of 13 SBG and 17 Bx from normal native SB as controls (ct) were analyzed by flow cytometry for ILC surface markers, intracellular IFNg, IL17 and IL22 and cytotoxicity. CD3- cells in SBG expressed CD56 (55% vs 37.1% in ct; p=0.001) and NKp44 (grafts=46.8%, ct=32.1%; p=0.035), both features of NK as well as subsets of ILC1 and ILC3. A similar proportion of grafts innate IEL expressed IL23R, (grafts=40.5%, ct=17.4%; p<0.001), which is absent in NK, suggesting that most SBG CD103+CD3- cells expressing CD56 and/or NKp44 were also IL23R+ and were then mostly ILC3. 35% in SBG vs 10% in SB ct innate IEL expressed CCR6 (p<0.001), also characteristic of some ILC1 and all ILC3 cells. However, less SBG CD3- IEL expressed the ILC3 marker RORC (10% vs 6.2% in ct; p=0.002), and there were less CD127+ ILCs in grafts than in ct (14.2% vs 39% respectively; p<0.001). Ex-vivo and after in vitro activation with PMA/Ionomycin, SBG IELs CD3- cells produced significantly higher IFNg and IL22, signature cytokines of ILC1 and ILC3 respectively. Remarkably, a double IFNg+IL22+ population was observed in SBG, virtually absent in native SB and in ct or recipients peripheral blood NK cells (PB-NK). Some production of ILC3-characteristic IL17 was recorded in a subset of SBG T IELs, but not in ILCs or PB-NK. EpCAM-depleted SBG IEL showed significantly higher cytotoxicity towards K562 cells than ct SB IEL, although Granzyme B expression was similar. Cytotoxicity was similar in recipients or ct PB-NK cells suggesting independence from immunosuppressive therapy. In conclusion, ILCs predominating in SBG show some features of ILC1/NK (CD56+, NKp44+, CD127-, cytotoxic activity and IFNg) and ILC3 (CD56+, NKp44+, IL23R+, CCR6+, RORC+/- and IL22). ILCs producing both IFNg and IL22 suggest that innate cells with an intermediate ILC1-ILC3 phenotype may exist. Viability of SBG probably depends on the balance among pro-inflammatory and homeostatic roles of these ILCs subsets.

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