Hobit confers tissue-dependent programs to type 1 innate lymphoid cells

Abstract

Identification of type 1 innate lymphoid cells (ILC1s) has been problematic. The transcription factor Hobit encoded by Zfp683 has been proposed as a major driver of ILC1 programs. Using Zfp683 reporter mice, we showed that correlation of Hobit expression with ILC1s is tissue- and context-dependent. In liver and intestinal mucosa, Zfp683 expression correlated well with ILC1s; in salivary glands, Zfp683 was coexpressed with the natural killer (NK) master transcription factors Eomes and TCF1 in a unique cell population, which we call ILC1-like NK cells; during viral infection, Zfp683 was induced in conventional NK cells of spleen and liver. The impact of Zfp683 deletion on ILC1s and NK cells was also multifaceted, including a marked decrease in granzyme- and interferon-gamma (IFNγ)-producing ILC1s in the liver, slightly fewer ILC1s and more Eomes⁺ TCF1⁺ ILC1-like NK cells in salivary glands, and only reduced production of granzyme B by ILC1 in the intestinal mucosa. NK cell-mediated control of viral infection was unaffected. We conclude that Hobit has two major impacts on ILC1s: It sustains liver ILC1 numbers, while promoting ILC1 functional maturation in other tissues by controlling TCF1, Eomes, and granzyme expression.

Keywords: Eomes; Hobit; innate lymphoid cells; liver; natural killer cells.

Fig. 1.

Zfp683 and Eomes define ILC1s and NK cells across tissues. (A) Schematic of targeting construct. (B) Representative flow cytometric dot plots and quantification of Zfp683⁺ cells among live, CD45⁺Lin⁻NK1.1⁺CD49a⁺, CD49b⁺, or CD127⁺ cells in the indicated organs. Single-cell suspensions were obtained from heterozygous Zfp683^Red mice. IELs, intraepithelial lymphocytes; SG, salivary gland; siLP, small intestinal lamina propria. Data are representative of two independent experiments (n = 3 or 4). Data represent mean ± SEM. (C) Expression of Eomes and Zfp683 in single-cell suspensions from different organs of Zfp683^Red heterozygous mice, and their quantification among NK1.1⁺ cells gated as in B. DP, double-positive; SP, single positive. Data represent mean ± SEM. Data are representative of two independent experiments (n = 3 or 4).

Fig. 2.

scRNA-seq delineates previously unappreciated ILC1 and NK cell subpopulations. (A) UMAP plot of 17,660 CD3⁻NK1.1⁺ cells from multiple tissues. Each cluster was annotated based on differentially expressed genes. (B) UMAP plots of representative selected genes that discriminate among the identified clusters. (C) UMAP plots of CD3⁻NK1.1⁺ cells from the indicated tissues.

Fig. 3.

CD127 expression identifies at least two liver ILC1 subsets. (A) Representative flow cytometric gating strategy for liver NK cell and ILC1 subsets and expression of CD127 on CD49a⁺CD49b⁻ ILC1s. (B) Representative flow cytometric dot plots and quantification of IFNγ and granzyme B expression in the indicated populations. It should be noted that liver NK cells include both iNK and mNK cells, which produce distinct amounts of IFNγ; therefore, the values of IFNγ depicted represent an average of the entire NK population. (C) Representative flow cytometric histogram plots and quantification of TCF1 protein expression in the indicated populations. (D) Representative flow cytometric dot plots showing expression of CD127 on RORγt fate-map (RORγtFM) liver CD49a⁺ ILC1s. (E) UMAP plots and cluster representation among CD3⁻NK1.1⁺ cells from wild-type or Zfp683^fl/fl Ncr1^Cre mice. (F) Representative flow cytometric dot plots and total cell counts of the indicated populations from wild-type or Zfp683^fl/fl Ncr1^Cremice. (G) Representative flow cytometric dot plots and quantification showing the expression of IFNγ and granzyme B in the indicated populations from wild-type or Zfp683^fl/fl Ncr1^Cre mice. (H and I) UMAP plots (H) and cluster proportion (I) of liver ILC1s from wild-type or Zfp683^fl/fl Ncr1^Cre mice. Data from A–D, F, and G are representative of two independent experiments (n = 4). Data represent mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001; n.s., not significant. MFI, mean fluorescence intensity; WT, wild type.

Fig. 4.

Hobit differentially regulates expression of granzyme B, TCF1, and Eomes in salivary gland and lamina propria ILC1s. (A and B) UMAP plots (A) and cluster representation (B) of salivary gland CD3⁻NK1.1⁺ cells from wild-type or Zfp683^fl/fl Ncr1^Cre mice. (C) Representative flow cytometric dot plots and quantification of Eomes^hi/lo and TCF1 expression cells among NK1.1⁺ cells in salivary glands from wild-type or Zfp683^fl/fl Ncr1^Cre mice. (D) Violin plots of granzyme B expression levels in each cluster of salivary gland CD3⁻NK1.1⁺ cells. (E) Representative flow cytometric dot plots and quantification of IFNγ and granzyme B in the indicated NK1.1⁺ populations of salivary glands from wild-type or Zfp683^fl/fl Ncr1^Cre mice. (F and G) UMAP plots (F) and cluster representation (G) in lamina propria CD3⁻NK1.1⁺ cells from wild-type or Zfp683^fl/fl Ncr1^Cre mice. (H) Representative flow cytometric dot plots showing expression of Eomes and TCF1 and quantification of absolute numbers of ILC1s and NK cells and of TCF1 expression in the indicated populations of lamina propria NK1.1⁺ cells from wild-type or Zfp683^fl/fl Ncr1^Cre mice. (I) Violin plots of granzyme B expression levels in each cluster of intestinal lamina propria CD3⁻NK1.1⁺ cells. (J) Representative flow cytometric dot plots and quantification of IFNγ and granzyme B in the indicated lamina propria NK1.1⁺ populations from wild-type or Zfp683^fl/fl Ncr1^Cre mice. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; n.s., not significant. (C, E, H, and J) Data are representative of two independent experiments (n = 4). (E and J) NK cells and ILC1s were stimulated with IL-12 and IL-18. All flow cytometric plots were pregated with the indicated markers. Data represent mean ± SEM.

Fig. 5.

NK cells and ILC1s are independent lineages, but a small portion of ILC1s convert to ILC3s. (A and B) Representative flow cytometric dot plots (A) and quantification (B) of Hobit-expressing (Zfp683^Red) versus Hobit fate-map (Zfp683^FM) among NK1.1⁺ cells in different organs. Single-cell suspensions were obtained from heterozygous Zfp683^Red mice. (C) Representative flow cytometric dot plots showing expression of Hobit (Zfp683^Red), Eomes, and RORγt among NKp46⁺ cells of the small intestinal lamina propria, which include ILC1s, ILC3s, and NK cells. (D and E) Representative flow cytometric dot plots (D) and quantification (E) showing expression of Hobit fate-map (Zfp683^FM) cells coexpressing RORγt or Zfp683. Data are representative of at least two independent experiments (n = 3 or 4). Data represent mean ± SEM.

Fig. 6.

MCMV infection induces Hobit expression in NK cells. (A and B) Representative flow cytometric dot plots (A) and quantification (B) of Hobit-expressing (Zfp683^Red) splenic NK cells at different time points after MCMV i.p. infection. (C and D) Representative dot plots of Hobit-expressing (Zfp683^Red) and Eomes-expressing liver ILC1s and NK cells (C) and their quantification (D) at the indicated time points after MCMV i.p. infection in heterozygous Zfp683^Red mice. (E) Absolute cell numbers of the indicated populations in the liver at the indicated time points post–MCMV i.p. infection. (F and G) Representative flow cytometric dot plots (F) and absolute cell counts (G) of Hobit-expressing (Zfp683^Red) or Hobit fate map (Zfp683^FM) among liver NK1.1⁺ cells at 1.5 d post–MCMV infection. (H and I) Representative flow cytometric dot plots (H) and quantification (I) of Hobit-expressing (Zfp683^Red) in splenic NK1.1⁺ cells after 72 h in culture with the indicated cytokines. Data are representative of at least two independent experiments (n = 5 to 8). Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n.s., not significant.