IL-7 and IL-15 independently program the differentiation of intestinal CD3-NKp46+ cell subsets from Id2-dependent precursors

Abstract

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.

Figure 1.

Normal development of splenic and intestinal CD3⁻NKp46⁺ cell subsets requires Id2. (A) NK1.1 versus CD127 expression on gated CD3⁻NKp46⁺ splenocytes and small intestinal (SI) LPLs of WT and Id2^−/− mice. Subset frequencies are indicated in one representative experiment out of six performed. (B) RORγt expression in intestinal CD3⁻NKp46⁺ subsets in WT (bold line) and Id2^−/− mice (shaded histogram). The dotted line depicts staining of CD3⁻NKp46⁺ cells from Rorc^−/− mice. (C) IL-23–induced IL-22 expression in intestinal CD3⁻NKp46⁺CD127⁺ cells from WT and Id2^−/− mice. Results indicate frequencies from one out of two independent experiments.

Figure 2.

Perturbed development of intestinal CD3⁻GFP⁺ cell subsets in Ncr1^GFP/+ mice that overexpress IL-15 in the intestinal epithelium. (A) NK1.1 versus CD127 expression on gated CD3⁻GFP⁺ cells from small intestinal (SI) LPL and intraepithelial (IEL) compartments. Subset frequencies are indicated. Representative results from one out of six independent experiments are shown. (B) Absolute numbers of total CD3⁻NKp46⁺ cells and CD127/NK1.1 subsets from small intestinal LPLs in WT and IL-15 Tg mice (n = 5–8 mice of each genotype analyzed). Only CD127⁻NK1.1⁺ cells were significantly increased in IL-15 Tg mice. Values are presented as means ± SD. (C) RORγt expression in intestinal CD3⁻NKp46⁺ subsets in WT (bold line) and IL-15 Tg mice (shaded histogram). The dotted line depicts staining of CD3⁻NKp46⁺ cells from Rorc^−/− mice. (D) IL-22 production from intestinal CD3⁻NKp46⁺ subsets in WT and IL-15 Tg mice. Results indicate frequencies from one out of three independent experiments.

Figure 3.

Altered development of intestinal CD3⁻NKp46⁺ cells in Il7^−/− mice. (A) NK1.1 versus CD127 expression on gated CD3⁻NKp46⁺ splenocytes and small intestinal (SI) LPLs of WT versus Il7^−/− mice. Subset frequencies are indicated in one representative experiment out of six performed. (B) Absolute numbers of total CD3⁻NKp46⁺ cells and CD127/NK1.1 subsets from small intestinal LPLs in WT and Il7^−/− mice (n = 6 mice of each genotype analyzed). Significant differences are indicated. Values are presented as means ± SD. (C) RORγt expression in the indicated intestinal CD3⁻NKp46⁺ subsets in WT (bold line) and Il7^−/− mice (shaded histogram). The dotted line depicts staining of CD3⁻NKp46⁺ cells from Rorc^−/− mice. (D) IL-22 production from intestinal CD3⁻NKp46⁺ subsets in WT and Il7^−/− mice. Results indicate frequencies from one out of three independent experiments.

Figure 4.

Fate-mapping analysis of Rorc-expressing precursors. (A) Frequency of YFP⁺ cells in gated CD3⁻NKp46⁺ cells from small intestinal (SI) LPLs and intraepithelial (IEL) cells. One representative experiment out of five is shown. Means of 51 ± 18% and 18 ± 12% YFP⁺ cells were detected in the LPL and intraepithelial compartments, respectively. (B) NK1.1 versus CD127 expression on YFP⁺ versus YFP⁻ subsets of intestinal CD3⁻NKp46⁺ small intestinal LPL and intraepithelial cells. (C) Frequency of YFP⁺ cells in gated CD3⁻NK1.1⁺ cells from the indicated organs/tissues. Results indicate frequencies from one representative experiment out of five performed.