ILC3-Derived OX40L Is Essential For Homeostasis Of Intestinal Tregs

OX40L is a member of tumor necrosis factor ligand superfamily?TNFSF?,and has been reported to be expressed by splenic lymphoid tissue inducer?Lti?cells,a subset of the group 3 innate lymphoid cells?ILC3s?.Whether OX40L is expressed on intestinal ILC3s and the molecular regulation of OX40L expression in ILC3s are unknown.Here,through flow cytometry,we showed ILC3s marked as an OX40L^highigh population among all intestinal lamina proprial leukocytes?LPLs?and were the dominant source for OX40L in Rag1^–/–mice.All ILC3 subsets expressed OX40L,and NCR^–ILC3s were the most abundant source of OX40L.OX40L expression on intestinal ILC3s of wildtype mice was much lower than in Rag1^–/–mice,suggesting a suppressive role of adaptive immune system on ILC3s.We transplanted different T cell subsets to Rag1^–/–mice and found that CD4⁺T cells suppressed the expression of OX40L on ILC3s.Through the co-culture of CD4⁺T cells and ILC3s,we further demonstrated that CD4⁺T cells suppressed OX40L expression on ILC3s through OX40.OX40L expression on large intestinal ILC3s was gradually increased with age,suggesting that environmental factors might induce the expression of OX40L.We induced colitis using DSS and anti-CD40 in Rag1^–/–mice and found that OX40L expression on large intestinal ILC3s was increased under inflammatory conditions.Consistently,significantly higher level of OX40L expression could be detected in ILC3s from conventional mice compared to mice housed in SPF?specific pathogen free?conditions.However,broad-spectrum antibiotics treatment failed to bring down OX40L expression on ILC3s,suggesting that inflammatory cues triggered by viral or fungal factors might sustain OX40L expression in ILC3s.We further found that inflammatory factor TL1A and Poly?I:C?could promote OX40L expression on ILC3s both in vitro and in vivo.Through the in vitro assay of the purified intestinal ILC3s,we demonstrated that Poly?I:C?promoted OX40L expression in a cell-intrinsic manner independent of TL1A.To determine the key downstream regulator of OX40L in ILC3s,we screened the inhibitors of a series of key signal regulators and transcriptional factors.We found that the STAT5 inhibitor,IQDMA,could effectively suppress the OX40L expression on ILC3s.IL-7 stimulated STAT5 phosphorylation in ILC3s and promoted OX40L expression on ILC3s,further proving that STAT5 regulated the expression of OX40L on intestinal ILC3s.Intestinal regulatory T cells?Tregs?constitutively express OX40.It has been widely reported that OX40 signaling was important for the expasion,survival and memory of Tregs.Whether ILC3s interact with Tregs through OX40L has not been reported so far.We transferred Tregs to Rag1^–/–Rorc^gfp/gfp mice and demonstrated that ILC3s were essential for the homeostatic expansion of intestinal Tregs.Through the construction of bone marrow chimeric mice in which OX40L was specifically deficient on ILC3s,we further demonstrated that ILC3-derived OX40L was essential for the homeostatic expansion of intestinal Tregs.Finally,we conducted immunofluorescence staining of intestinal frozen sections.Colocalization of Tregs and ILC3s was found in the intestinal cryptopatches,suggesting a direct interaction of Tregs and ILC3s,possibly through OX40L-OX40 interaction.In conclusion,we found ILC3s were the major source for OX40L among intestinal leukocytes.The expression of OX40L in intestinal ILC3s was gradually increased after birth and NCR^–ILC3s were the most abundant source of OX40L.The expression of OX40L on intestinal ILC3s was triggered by inflammation and suppressed by CD4⁺T cells.Moreover,we demonstrated that IL-7-STAT5 signaling sustained OX40L expression by intestinal ILC3s.Finally,we showed that ILC3-derived OX40L was essential for Treg homeostatic expansion in the intestine,and further found the colocalization of ILC3s and Tregs in cryptopatches.Our study reveals a supportive role of ILC3s on Tregs in the intestine through OX40L-OX40 interaction.