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Distinct requirements for T-bet in gut innate lymphoid cells.

Sciumé G, Hirahara K, Takahashi H, Laurence A, Villarino AV, Singleton KL, Spencer SP, Wilhelm C, Poholek AC, Vahedi G, Kanno Y, Belkaid Y, O'Shea JJ - J. Exp. Med. (2012)

Bottom Line: The residual NKp46+ ILC22 present in Tbx21(-/-) mice showed a marked reduction of Rorγt expression and impairment in IL-22 production.Bone marrow chimera experiments revealed a cell-intrinsic requirement for T-bet in these subsets and competitive reconstitution experiments revealed roles for T-bet in multiple ILC subsets.Thus, T-bet has a general importance for ILC in the gut and plays a selective and critical role in the generation of NKp46+ ILC22.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lymphocyte Cell Biology Section, Molecular Immunology and Inflammation Branch, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Interleukin (IL)-22-producing innate lymphoid cells (ILCs; ILC22) comprise a heterogeneous population of cells that are dependent on the transcription factor retinoid-related orphan γt (RORγt) and are critical for barrier function of the intestinal mucosa. A distinct ILC22 subset expresses the natural cytotoxicity receptor NKp46 (NKp46+ ILC22); however, the factors that contribute to the generation of this population versus other subsets are largely unknown. Herein, we show that T-bet (encoded by Tbx21) was highly expressed in NKp46+ ILC22, a feature shared by all NKp46+ cells present in the intestine but not by other IL-22-producing populations. Accordingly, the absence of T-bet resulted in loss of NKp46+ ILC22 in the intestinal lamina propria. The residual NKp46+ ILC22 present in Tbx21(-/-) mice showed a marked reduction of Rorγt expression and impairment in IL-22 production. Generation and functions of gut NK1.1+ cells were also altered. Bone marrow chimera experiments revealed a cell-intrinsic requirement for T-bet in these subsets and competitive reconstitution experiments revealed roles for T-bet in multiple ILC subsets. Thus, T-bet has a general importance for ILC in the gut and plays a selective and critical role in the generation of NKp46+ ILC22.

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NKp46+ ILC22 and CD127+ NK cells are dramatically reduced in Tbx21−/− mice. Lymphocytes were isolated from siLP of WT and Tbx21−/− mice and analyzed by flow cytometry. (A) The left panel shows representative dot plots and the right panels show absolute number of CD127+ and CD127− CD3ε−NK1.1+ populations (mean ± SD). (B) Representative dot plots and absolute number of the different ILC22 populations. Values represent mean ± SD of each indicated population (WT, five mice; Tbx21−/−, six mice). Four independent experiments were performed. Asterisks denote P < 0.05. (C) Representative dot plots of Rorγt+ ILC populations (according CD127, CD117, CD4, and NKp46 expression) and absolute number (mean ± SD) of Rorγt+CD127lowCD117low ILC in WT and Tbx21−/− mice (WT, three mice; Tbx21−/−, three mice). Two independent experiments were performed. Asterisk denotes P < 0.05.
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fig2: NKp46+ ILC22 and CD127+ NK cells are dramatically reduced in Tbx21−/− mice. Lymphocytes were isolated from siLP of WT and Tbx21−/− mice and analyzed by flow cytometry. (A) The left panel shows representative dot plots and the right panels show absolute number of CD127+ and CD127− CD3ε−NK1.1+ populations (mean ± SD). (B) Representative dot plots and absolute number of the different ILC22 populations. Values represent mean ± SD of each indicated population (WT, five mice; Tbx21−/−, six mice). Four independent experiments were performed. Asterisks denote P < 0.05. (C) Representative dot plots of Rorγt+ ILC populations (according CD127, CD117, CD4, and NKp46 expression) and absolute number (mean ± SD) of Rorγt+CD127lowCD117low ILC in WT and Tbx21−/− mice (WT, three mice; Tbx21−/−, three mice). Two independent experiments were performed. Asterisk denotes P < 0.05.

Mentions: To elucidate the potential impact of T-bet in these ILC subsets, we assessed the various populations of gut innate lymphocytes in Tbx21−/− mice by applying the same gating strategy used in Fig. 1. Despite the homogeneous expression of T-bet among all CD3ε−NK1.1+ cells (CD127− and CD127+), Tbx21−/− mice showed a dramatic and selective reduction of CD127+ NK cells. In contrast, there were no significant differences in CD127− NK cells, compared with WT mice (Fig. 2 A). When we analyzed CD127+Rorγt+ ILC populations, we found no difference in the number of CD4− ILC22 or LTi4 cells. However, in agreement with the pattern of T-bet expression, we did note a striking and selective decrease in the number of NKp46+ ILC22 cells in T-bet–deficient mice (Fig. 2 B). It was possible though that T-bet was simply regulating NKp46 expression on this subset. We attacked this problem in two ways. First, we found that NKp46 expression on conventional NK cells was not affected by the absence of T-bet (unpublished data). Second, we enumerated ILC22 cells using markers independent of NKp46+. ILC22 have been shown to be enriched in the Rorγt+CD127lowCD117low population of lamina propria cells (Sawa et al., 2010 and Fig. 2 C). We observed a significant reduced proportion of this population of cells in Tbx21−/− mice, independent of reliance on NKp46 expression (Fig. 2 C). In addition, Tbx21−/− mice had normal numbers of CD45+Thy1.2+lin− cells, which contain ILC2 and lacked T-bet expression (unpublished data). Collectively, these data argue that indeed, NKp46+ ILC22 cells are preferentially affected by the loss of T-bet.


Distinct requirements for T-bet in gut innate lymphoid cells.

Sciumé G, Hirahara K, Takahashi H, Laurence A, Villarino AV, Singleton KL, Spencer SP, Wilhelm C, Poholek AC, Vahedi G, Kanno Y, Belkaid Y, O'Shea JJ - J. Exp. Med. (2012)

NKp46+ ILC22 and CD127+ NK cells are dramatically reduced in Tbx21−/− mice. Lymphocytes were isolated from siLP of WT and Tbx21−/− mice and analyzed by flow cytometry. (A) The left panel shows representative dot plots and the right panels show absolute number of CD127+ and CD127− CD3ε−NK1.1+ populations (mean ± SD). (B) Representative dot plots and absolute number of the different ILC22 populations. Values represent mean ± SD of each indicated population (WT, five mice; Tbx21−/−, six mice). Four independent experiments were performed. Asterisks denote P < 0.05. (C) Representative dot plots of Rorγt+ ILC populations (according CD127, CD117, CD4, and NKp46 expression) and absolute number (mean ± SD) of Rorγt+CD127lowCD117low ILC in WT and Tbx21−/− mice (WT, three mice; Tbx21−/−, three mice). Two independent experiments were performed. Asterisk denotes P < 0.05.
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fig2: NKp46+ ILC22 and CD127+ NK cells are dramatically reduced in Tbx21−/− mice. Lymphocytes were isolated from siLP of WT and Tbx21−/− mice and analyzed by flow cytometry. (A) The left panel shows representative dot plots and the right panels show absolute number of CD127+ and CD127− CD3ε−NK1.1+ populations (mean ± SD). (B) Representative dot plots and absolute number of the different ILC22 populations. Values represent mean ± SD of each indicated population (WT, five mice; Tbx21−/−, six mice). Four independent experiments were performed. Asterisks denote P < 0.05. (C) Representative dot plots of Rorγt+ ILC populations (according CD127, CD117, CD4, and NKp46 expression) and absolute number (mean ± SD) of Rorγt+CD127lowCD117low ILC in WT and Tbx21−/− mice (WT, three mice; Tbx21−/−, three mice). Two independent experiments were performed. Asterisk denotes P < 0.05.
Mentions: To elucidate the potential impact of T-bet in these ILC subsets, we assessed the various populations of gut innate lymphocytes in Tbx21−/− mice by applying the same gating strategy used in Fig. 1. Despite the homogeneous expression of T-bet among all CD3ε−NK1.1+ cells (CD127− and CD127+), Tbx21−/− mice showed a dramatic and selective reduction of CD127+ NK cells. In contrast, there were no significant differences in CD127− NK cells, compared with WT mice (Fig. 2 A). When we analyzed CD127+Rorγt+ ILC populations, we found no difference in the number of CD4− ILC22 or LTi4 cells. However, in agreement with the pattern of T-bet expression, we did note a striking and selective decrease in the number of NKp46+ ILC22 cells in T-bet–deficient mice (Fig. 2 B). It was possible though that T-bet was simply regulating NKp46 expression on this subset. We attacked this problem in two ways. First, we found that NKp46 expression on conventional NK cells was not affected by the absence of T-bet (unpublished data). Second, we enumerated ILC22 cells using markers independent of NKp46+. ILC22 have been shown to be enriched in the Rorγt+CD127lowCD117low population of lamina propria cells (Sawa et al., 2010 and Fig. 2 C). We observed a significant reduced proportion of this population of cells in Tbx21−/− mice, independent of reliance on NKp46 expression (Fig. 2 C). In addition, Tbx21−/− mice had normal numbers of CD45+Thy1.2+lin− cells, which contain ILC2 and lacked T-bet expression (unpublished data). Collectively, these data argue that indeed, NKp46+ ILC22 cells are preferentially affected by the loss of T-bet.

Bottom Line: The residual NKp46+ ILC22 present in Tbx21(-/-) mice showed a marked reduction of Rorγt expression and impairment in IL-22 production.Bone marrow chimera experiments revealed a cell-intrinsic requirement for T-bet in these subsets and competitive reconstitution experiments revealed roles for T-bet in multiple ILC subsets.Thus, T-bet has a general importance for ILC in the gut and plays a selective and critical role in the generation of NKp46+ ILC22.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lymphocyte Cell Biology Section, Molecular Immunology and Inflammation Branch, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Interleukin (IL)-22-producing innate lymphoid cells (ILCs; ILC22) comprise a heterogeneous population of cells that are dependent on the transcription factor retinoid-related orphan γt (RORγt) and are critical for barrier function of the intestinal mucosa. A distinct ILC22 subset expresses the natural cytotoxicity receptor NKp46 (NKp46+ ILC22); however, the factors that contribute to the generation of this population versus other subsets are largely unknown. Herein, we show that T-bet (encoded by Tbx21) was highly expressed in NKp46+ ILC22, a feature shared by all NKp46+ cells present in the intestine but not by other IL-22-producing populations. Accordingly, the absence of T-bet resulted in loss of NKp46+ ILC22 in the intestinal lamina propria. The residual NKp46+ ILC22 present in Tbx21(-/-) mice showed a marked reduction of Rorγt expression and impairment in IL-22 production. Generation and functions of gut NK1.1+ cells were also altered. Bone marrow chimera experiments revealed a cell-intrinsic requirement for T-bet in these subsets and competitive reconstitution experiments revealed roles for T-bet in multiple ILC subsets. Thus, T-bet has a general importance for ILC in the gut and plays a selective and critical role in the generation of NKp46+ ILC22.

Show MeSH
Related in: MedlinePlus