Natural killer (NK) cells are innate lymphocytes with spontaneous antitumor activity, and they produce interferon-gamma (IFN-gamma) that primes immune responses. Whereas T helper cell subsets differentiate from naive T cells via specific transcription factors, evidence for NK cell diversification is limited. In this report, we characterized intestinal lymphocytes expressing the NK cell natural cytotoxicity receptor NKp46. Gut NKp46+ cells were distinguished from classical NK cells by limited IFN-gamma production and absence of perforin, whereas several subsets expressed the nuclear hormone receptor retinoic acid receptor-related orphan receptor t (RORgammat) and interleukin-22 (IL-22). Intestinal NKp46+IL-22+ cells were generated via a local process that was conditioned by commensal bacteria and required RORgammat. Mice lacking IL-22-producing NKp46+ cells showed heightened susceptibility to the pathogen Citrobacter rodentium, consistent with a role for intestinal NKp46+ cells in immune protection. RORgammat-driven diversification of intestinal NKp46+ cells thereby specifies an innate cellular defense mechanism that operates at mucosal surfaces.

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Lymphoid tissue-inducer (LTi) cells initiate the development of lymphoid tissues through the activation of local stromal cells in a process similar to inflammation. LTi cells express the nuclear hormone receptor RORγt, which also directs the expression of the proinflammatory cytokine interleukin-17 in T cells. We show here that LTi cells are part of a larger family of proinflammatory RORγt(+) innate lymphoid cells (ILCs) that differentiate from distinct fetal liver RORγt(+) precursors. The fate of RORγt(+) ILCs is determined by mouse age, and after birth, favors the generation of cells involved in intestinal homeostasis and defense. Contrary to RORγt(+) T cells, however, RORγt(+) ILCs develop in the absence of microbiota. Our study indicates that RORγt(+) ILCs evolve to preempt intestinal colonization by microbial symbionts.

The natural cytotoxicity receptor NKp46 (encoded by Ncr1) was recently shown to identify a subset of noncytotoxic, Rag-independent gut lymphocytes that express the transcription factor Rorc, produce interleukin (IL)-22, and provide innate immune protection at the intestinal mucosa. Intestinal CD3−NKp46+ cells are phenotypically heterogeneous, comprising a minority subset that resembles classical mature splenic natural killer (NK) cells (NK1.1+, Ly49+) but also a large CD127+NK1.1− subset of lymphoid tissue inducer (LTi)–like Rorc+ cells that has been proposed to include NK cell precursors. We investigated the developmental relationships between these intestinal CD3−NKp46+ subsets. Gut CD3−NKp46+ cells were related to LTi and NK cells in requiring the transcriptional inhibitor Id2 for normal development. Overexpression of IL-15 in intestinal epithelial cells expanded NK1.1+ cells within the gut but had no effect on absolute numbers of the CD127+NK1.1−Rorc+ subset of CD3−NKp46+ cells. In contrast, IL-7 deficiency strongly reduced the overall numbers of CD3−NKp46+NK1.1− cells that express Rorc and produce IL-22 but failed to restrict homeostasis of classical intestinal NK1.1+ cells. Finally, in vivo fate-mapping experiments demonstrated that intestinal NK1.1+CD127− cells are not the progeny of Rorc-expressing progenitors, indicating that CD127+NK1.1−Rorc+ cells are not canonical NK cell precursors. These studies highlight the independent cytokine regulation of functionally diverse intestinal NKp46+ cell subsets.

Lymphoid tissue inducer cells are members of an emerging family of innate lymphoid cells (ILC). Although these cells were originally reported to produce cytokines such as interleukin-17 (IL-17) and IL-22, we demonstrate here that human CD127(+)RORC(+) and CD56(+)CD127(+) LTi-like ILC also express IL-2, IL-5, and IL-13 after activation with physiologic stimuli such as common γ-chain cytokines, Toll-like receptor (TLR) 2 ligands, or IL-23. Whereas TLR2 signaling induced IL-5, IL-13, and IL-22 expression in a nuclear factor κB (NF-κB)-dependent manner, IL-23 costimulation induced only IL-22 production. CD127(+) LTi-like ILC displayed clonal heterogeneity for IL-13 and IL-5 production, suggesting in vivo polarization. Finally, we identified a role for autocrine IL-2 signaling in mediating the effects of TLR2 stimulation on CD56(+)CD127(+) and CD127(+) LTi-like ILC. These results indicate that human LTi-like ILC can directly sense bacterial components and unravel a previously unrecognized functional heterogeneity among this important population of innate lymphoid cells.

The transcription factor Gata3 is required for the generation of group 3 innate lymphoid cells (ILC3) that protect mucosal surfaces.

Interleukin-22 (IL-22) plays a critical role in mucosal defense, although the molecular mechanisms that ensure IL-22 tissue distribution remain poorly understood. We show that the CXCL16-CXCR6 chemokine-chemokine receptor axis regulated group 3 innate lymphoid cell (ILC3) diversity and function. CXCL16 was constitutively expressed by CX3CR1(+) intestinal dendritic cells (DCs) and coexpressed with IL-23 after Citrobacter rodentium infection. Intestinal ILC3s expressed CXCR6 and its ablation generated a selective loss of the NKp46(+) ILC3 subset, a depletion of intestinal IL-22, and the inability to control C. rodentium infection. CD4(+) ILC3s were unaffected by CXCR6 deficiency and remained clustered within lymphoid follicles. In contrast, the lamina propria of Cxcr6(-/-) mice was devoid of ILC3s. The loss of ILC3-dependent IL-22 epithelial stimulation reduced antimicrobial peptide expression that explained the sensitivity of Cxcr6(-/-) mice to C. rodentium. Our results delineate a critical CXCL16-CXCR6 crosstalk that coordinates the intestinal topography of IL-22 secretion required for mucosal defense.

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