A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity

Abstract

Natural killer (NK) cells are classically viewed as lymphocytes that provide innate surveillance against virally infected cells and tumour cells through the release of cytolytic mediators and interferon (IFN)-gamma. In humans, blood CD56(dim) NK cells specialize in the lysis of cell targets. In the lymph nodes, CD56(bright) NK cells secrete IFN-gamma cooperating with dendritic cells and T cells in the generation of adaptive responses. Here we report the characterization of a human NK cell subset located in mucosa-associated lymphoid tissues, such as tonsils and Peyer's patches, which is hard-wired to secrete interleukin (IL)-22, IL-26 and leukaemia inhibitory factor. These NK cells, which we refer to as NK-22 cells, are triggered by acute exposure to IL-23. In vitro, NK-22-secreted cytokines stimulate epithelial cells to secrete IL-10, proliferate and express a variety of mitogenic and anti-apoptotic molecules. NK-22 cells are also found in mouse mucosa-associated lymphoid tissues and appear in the small intestine lamina propria during bacterial infection, suggesting that NK-22 cells provide an innate source of IL-22 that may help constrain inflammation and protect mucosal sites.

Figure 1. NKp44⁺ NK cells are prominently found within MALT

a, Immunohistochemical analysis of tonsil sections with anti-NKp44 shows NKp44⁺ NK cells residing within the tonsil epithelium and the lamina propria. b, Double immunofluorescence confirms that NKp44⁺ cells (red) are either in the lamina propria, or within the surface epithelium in close contact with cytokeratin-5⁺ (green) epithelial cells (white arrowheads). c, At high power view, NKp44⁺ NK cells show a round to oval morphology and co-express CD56 (inset). d,e, Numerous NKp44⁺ NK cells are present in the dome region of ileum Peyer's patches (d) and in the luminal epithelium, dome and interfollicular regions of the appendix (e).

Figure 2. A subset of tonsil NKp44⁺ NK cells express CCR6, respond to CCL20 in vitro and in vivo, produce CCL20, and adhere to epithelial cells

a, Approximately 50% of NKp44⁺ NK cells express CCR6. b, Tonsil NK cells migrate in response to CCL20. Bars indicate the ratio between the percentage of input NKp44⁺ NK cells versus the percentage of migrated NKp44⁺ NK cells. c, PMA/ionomycin treated NKp44⁺ NK cells produce significantly more CCL20 than NKp44⁻ NK cells. d, e, A case of dermatitis with pathological LC accumulation shows extensive skin infiltration of NKp44⁺ NK cells as compared to a normal skin. Arrows indicate pseudo-Pautrier abscesses indicative of LC accumulation. f, Real time PCR shows that skin with LC accumulation produces more CCL20 than normal skin. g, NKp44⁺ NK cells express higher levels of CD96 than NKp44⁻ NK cells. h, A discrete subset of NKp44⁺ NK cells expresses CD103. i, NKp44⁺ NK cells firmly adhere to epithelial cells. The ratio of NKp44⁺ to NKp44⁻ cells within non-adherent and adherent CD3⁻CD56⁺ cells was determined by flow cytometry. j, The adhesiveness of NKp44⁺ NK cells to epithelial cells is consistent in different donors. All error bars indicate s.d. with n=3

Figure 3. Tonsil NKp44⁺ NK cells produce IL-22, IL-26 and LIF. IL-23 and TLR-activated monocytes trigger IL-22 secretion

a, IL-22 concentration in supernatants of NKp44⁺ and NKp44⁻ tonsil NK cells stimulated with PMA/ionomycin. b, Intracellular IL-22 and LIF content of tonsil NKp44⁺ cells and blood NK cells stimulated with PMA/ionomycin. c, NKp44⁺ NK cells express higher levels of IL-26 mRNA than NKp44⁻ NK cells. d-e, IL-23 triggers secretion of IL-22 (d, e) but not IL-17 (e) by NK-22 cells. MFI, mean fluorescence intensity. f, NKp44⁺ NK cells produce IL-22 in response to IL-23 and, to some extent, IL-15. NKp44⁻ NK cells release IFN-γ in response to IL-12 or IL-15. g, Monocytes stimulated with resiquimod and LPS induce IL-22 secretion in NK-22 cells. All error bars indicate s.d. with n=3

Figure 4. NK-22 cell-secreted cytokines stimulate epithelial cells to proliferate, release IL-10 and activate STAT1 and STAT3

a, Colo205 proliferation in response to supernatants derived from resting or IL-23-stimulated tonsil NK cells (left panel) or IL-22 (right panel). b, IL-10 secretion by Colo205 stimulated with supernatants from activated NKp44⁺ or NKp44⁻ tonsil NK cells or IL-22. c, STAT3 and STAT1 phosphorylation in Colo205 stimulated with supernatants of IL-23-activated NKp44⁺ NK cells (left panels, grey histograms), NKp44⁻ NK cells (left panels, empty histograms), IL-22 (right panels, grey histograms) or medium (right panels, empty histograms). As an additional control for STAT1 phosphorylation, cells were stimulated with IFN-γ (dotted line). All error bars indicate s.d. with n=3

Figure 5. Identification of mouse NK-22 cells

a, In vitro stimulation of Peyer’s patches cells with IL-23 induces production of IL-22 by NKp46⁺ NK cells. A gate was applied to exclude CD3⁺CD19⁺ cells; b, NKp46⁺ IL-22-producing cells are in part NK1.1⁻ and, to a lower degree, NK1.1⁺. c, NK-22 cell appear in the small intestine lamina propria of C. rodentium-infected mice. A large proportion (55-80% in different experiments) of cells producing IL-22 in the lamina propria express NKp46.