Type 3 Innate Lymphoid Cells Acquire a Cytotoxic Program upon Stimulation with Interleukins 12 and 15

Abstract

Type 3 innate lymphoid cells (ILCs) mediate tissue homeostasis and protection, primarily at mucosal sites. Their profile is tightly controlled by the tissue microenvironment and can shift toward production of the type 1 cytokine IFNγ in an inflammatory setting. Likely because ILC3 and natural killer (NK) cells are developmentally divergent innate lymphocyte lineages in mice, the plasticity of ILC3s with respect to cytotoxic function has been neglected. Since RORγt-expressing progenitors, however, contribute to NK cell generation in humans, we aimed to address this knowledge gap both in vitro and in vivo, using humanized mice. We could demonstrate that after exposure to IL-12 and IL-15, human ILC3s up-regulate expression of the master regulator of cytotoxic function Eomes and also the type 1 immunity associated transcription factor T-bet, albeit to a lesser extent. Transcriptional profiling and flow cytometric analysis revealed acquisition of key NK cell features like expression of CD94, NKG2A, NKG2C, CD56 and CD16 as well as enhanced responsiveness towards IL-2, IL-12 and IL-15. Functionally, expanded ILC3s gain the capacity to produce IFNγ among other NK cell-associated cytokines and chemokines. In addition they express cytolytic effector molecules such as granzymes and perforin and mount a cytotoxic response against the classical NK targets K562. They show slower and lower cytolytic activity compared to conventional NK cells, likely a result of a less potent activation or a distinct cytolytic mechanism. Importantly, we observed differences in cytotoxic potential between tonsil and spleen ILC3 subsets, which might indicate tissue-specific imprinting. In addition, a fraction of the ILC3s retains their phenotype despite the inflammatory cues, which suggests that not all ILC3s are primed to respond with differentiation to inflammation. In light of these findings and as helper CD4+ T cells can also switch on a cytotoxic program in symptomatic Epstein Barr virus (EBV) infection, we studied ILC3s in vivo under EBV-driven inflammatory conditions. A short-term exposure of autologous unmanipulated ILC3s in that setting seems to induce expression of CD94. However, transfer of IL-2 and IL-7 expanded ILC3s prior to virus inoculation had no consistent effects on EBV infection and pathogenesis, which though could be due to loss of the transferred cells.