The transcription factor GATA-3 controls cell fate and maintenance of type 2 innate lymphoid cells

Abstract

Innate lymphoid cells (ILCs) reside at mucosal surfaces and control immunity to intestinal infections. Type 2 innate lymphoid cells (ILC2s) produce cytokines such as IL-5 and IL-13, are required for immune defense against helminth infections, and are involved in the pathogenesis of airway hyperreactivity. Here, we have investigated the role of the transcription factor GATA-3 for ILC2 differentiation and maintenance. We showed that ILC2s and their lineage-specified bone marrow precursors (ILC2Ps), as identified here, were characterized by continuous high expression of GATA-3. Analysis of mice with temporary deletion of GATA-3 in all ILCs showed that GATA-3 was required for the differentiation and maintenance of ILC2s but not for RORγt(+) ILCs. Thus, our data demonstrate that GATA-3 is essential for ILC2 fate decisions and reveal similarities between the transcriptional programs controlling ILC and T helper cell fates.

Figure 1. ILC2 cells are GATA3-expressing innate lymphoid cells

(A) Expression of GATA3 by CD25⁺Sca1^hi (top) or CD127⁺Sca1^hi (bottom) lamina propria lymphocytes from the small intestine. Dot plots were gated on CD45⁺CD3⁻CD19⁻ lymphocytes. Numbers represent percentages of cells in the indicated gates. (B) Representative flow cytometry analyses of Sca1 and GATA3 expression (top) by CD45⁺CD3⁻CD19⁻ lymphocytes from the indicated organs. Histograms (bottom) represent staining with CD25 antibody (grey) or control Ig (open) by electronically gated GATA3^hiSca1^hi cells. Numbers represent percentage of cells in quadrant. (C) Lamina propria lymphocytes from the small intestine were co-stained with CD3, CD19, Sca1 and GATA3 antibodies and with antibodies specific for the indicated markers (grey) or isotype control Ab (open). Histograms are electronically gated on Sca1^hi GATA3^hi cells within the CD45⁺CD3⁻CD19⁻ population. (D,E) Intracellular cytokine staining of lamina propria lymphocytes from Gata3^Gfp/+ mice after 4h stimulation with PMA and ionomycin. (D) Dot plots are gated on CD3⁻CD19⁻ CD45⁺CD90⁺ lymphocytes. Numbers represent percentage of cells per quadrant. (E) Percentage (± SEM; n=5) of cytokine-producing GATA3^hi ILC with (black bars) and without stimulation (white bars). (F) Flow cytometry analysis of GATA3 and Sca1 expression by CD45⁺CD3⁻CD19⁻ cells from the intestinal lamina propria of the indicated mouse strains. (G) Absolute cell numbers of of GATA3^hiSca1^hi ILCs (± SD; n=4) in the lamina propria of the small intestine isolated from the indicated mouse strains. Data are representative of at least three independent experiments. See also Figure S1.

Figure 2. GATA3^high ILCs express KLRG1

(A) Coexpression of KLRG1 and CD25 or CD25 and GATA3 by electronically gated GATA3^hiSca1^hi (top) or KLRG1^hiSca1^hi (bottom) CD45⁺CD3⁻CD19⁻ lamina propria leukocytes. Numbers indicate percent of cells in the indicated regions. (B) Flow cytometry analysis of KLRG1 co-expression with the indicated cell surface markers and transcription factors by CD45⁺CD3⁻CD19⁻ lamina propria leukocytes. Numbers represent percentage of cells per quadrant. (C,D) Intracellular cytokine staining of lamina propria lymphocytes after 4h stimulation with PMA and ionomycin. (C) Dot plots are gated on CD45⁺CD3⁻CD19⁻CD90⁺ lymphocytes. Numbers represent percentage of cells per quadrant. (D) Percentage (± SEM; n=6) of cytokine-producing GATA3^hi ILC with (black bars) and without stimulation (white bars). (E) Lamina propria cells from mice at the indicated age were analyzed for GATA3 and RORγt expression (top). Dot plots are electronically gated on CD45⁺CD3⁻CD19⁻CD127⁺ cells. Numbers represent percentage of cells per quadrant. Histograms represent expression of KLRG1 and integrin α₄β₇ by electronically gated GATA3^hiRORγt⁻ cells. All data shown are representative of at least 3 independent experiments.

Figure 3. Identification of GATA3^hi ILCs in the bone marrow

(A,B) Expression of Id2 (GFP) (grey) by the indicated cell subsets obtained from spleen or intestinal lamina propria of Id2^Gfp/+ mice. Open histograms represent analysis of the same population from control mice (A). (B) Mean fluorescence intensity (MFI ± SEM; n=4) of Id2 (GFP) expression in comparison to control mice by the indicated cell subsets isolated from the intestinal lamina propria. (C) Id2 (GFP) and GATA3 (GFP) expression (grey) by the indicated bone marrow cell populations (HSC/MPP: Lin⁻CD127⁻Sca1⁺Kit⁺; CLP: Lin⁻CD127⁺CD135⁺Sca1^loKit^lo). Open histograms represent analysis of the same population from control mice. Numbers indicate percentage of positive cells. (D) Analyses of Sca1, Id2 (GFP) and GATA3 expression by Lin⁻ cells. The dot plots to the right show CD127 and CD25 expression by electronically gated Sca1^hiId2^hi (top) or Sca1^hiGATA3^hi cells (bottom). Numbers represent percentage of cells in the indicated gates. (E) Flow cytometry analysis of CD44 and CD90 expression (grey) by bone marrow LSI cells. Open histograms show staining with an isotype-matched control antibody. (F) The indicated mouse strains were analyzed for the presence of bone marrow Lin⁻ Sca1^hiGATA3^hi cells. Numbers represent percentage of cells in the indicated gates. Data are representative of at least three independent experiments. ** p ≤ 0.01, *** p ≤ 0.001. LSI: Lin⁻Sca1^hiId2^hi bone marrow cells. See also Figure S2.

Figure 4. Transcriptome analysis reveals close relationship between LSIG cells and ILC2

(A) Flow cytometry analysis of the indicated cell surface markers (grey) by bone marrow LSIG cells. Open histograms depict staining with isotype-matched control antibodies. (B,C) Quantitative RT-PCR analysis (± SEM; n=3) of expression of the indicated genes by LSIG cells (white bars) or ILC2 (black bars). (D) Percentage (± SEM; n=4) of IL-5-producing bone marrow LSIG cells (white bars) compared to ILC2 from the small intestine (black bars). (E) Hierarchical clustering of normalized microarray data of replicate RNA samples from the indicated cell subsets. Dendrogram was obtained by analyzing 911 out of 28,441 probesets. (F) Heat map representation of genes clustering with at least two fold differences in expression pattern across different cell subsets as assessed by k-means. Columns represent the indicated cell subsets in 4 or 5 biological replicates. Each row represents one examined gene. Hierarchical clustering revealed 10 different clusters as indicated. The color code at the bottom defines the expression intensity of each individual gene in all examined cell subsets. (G) Functional classification of the gene profiles shared by LSIG and ILC2 (i.e., ILC2/LSIG core cluster) and specific for ILC2 as assessed by k-means. Representative genes within these clusters are indicated. Signature gene expression profiles of the respective clusters used in the functional annotation are indicated above. Data are representative of three independent experiments. ** p ≤ 0.01. LSIG: Lin⁻ Sca1^hiId2^hiGATA3^hi bone marrow cells. See also Figure S3.

Figure 5. LSIG cells are lineage-specified progenitors of ILC2

(A) Highly purified LSIG cells from Gata3^Gfp/+ mice or Id2^Gfp/+ mice cells were cultured for 6 days on OP9 feeder cells in medium containing IL-7 and IL-15 and tested for the expression of NKp46 and GATA3 (left panel) or NK1.1 and Id2 (right panel). Numbers represent percentage of cells in quadrant. (B) Highly purified LSIG cells were cultured on OP9 feeder cells in the presence of the indicated cytokines. KLRG1 and GATA3 expression were determined after 7 days of culture. Numbers represent percentage of cells in quadrant. (C) Quantitative RT-PCR analysis of expression of the indicated genes by ILC2 (black bars), by LSIG cells cultured for 7 days with cytokines favoring differentiation of RORγt⁺ ILCs (IL-2, IL-7, IL-23, IL-1β) (white bars) or by RORγt⁺ ILCs (grey bars). (D) Percentage (± SD; n = 3) of KLRG1⁺ cells after 7 day culture of purified LSIG cells on OP9 feeder cells in the presence of the indicated cytokines. (E) Highly purified LSIG cells were cultured on OP9 feeder cells in the presence of the indicated cytokines. After 11 days of culture, IL-5 and IFN-γ production was determined by intracellular cytokine staining. Numbers represent percentage of cells per quadrant. (F) Percentage (± SD; n=3) of IL5⁺ cells after 7 day culture of purified LSIG cells on OP9 feeder cells in the presence of the indicated cytokines. (G) Highly purified bone marrow LSIG cells from Id2^Gfp/+ mice (H-2^b) were adoptively transferred into irradiated Rag2^−/−Il2rg^−/− hosts (H-2^d). Six weeks after transfer, donor-derived cells from the intestinal lamina propria were analyzed for the expression of the indicated markers. Numbers represent percentage of cells in the indicated gates. (H,I) The indicated numbers of highly purified bone marrow LSIG cells (H-2^b) or intestinal ILC2 (H-2^b) were adoptively transferred into irradiated Rag2^−/−Il2rg^−/− hosts (H-2^d). Four months after transfer, the fraction of donor-derived KLRG1⁺ ILC2 among lamina propria leukocytes was determined. As a measure of repopulation efficiency, the ratio (± SEM, n=3) of LSIG cell-derived to ILC2-derived KLRG1⁺ cells was determined (H). (J) Quantitative RT-PCR analysis of Rora expression by the indicated cell subsets from bone marrow or the small intestine. All experiments are representative of at least two independent experiments. LSIG: Lin⁻ Sca1^hiId2^hiGATA3^hi bone marrow cells. See also Figure S4.

Figure 6. Transfer of LSIG cells confers immunity to N. brasiliensis infection

(A-G) Highly purified LSIG cells were transferred into groups of Rag2^−/−Il2rg^−/− hosts. Four weeks after transfer, the indicated groups of mice (n=4-5) were infected with N. brasiliensis and analyzed on day 5 of infection. (A) Total intestinal helminth counts. (B) Percentage (± SD; n=3) of donor-derived (CD45.1⁺) ILC2 in the indicated organs at the day of infection (white bars) and at day 5 after N. brasiliensis infection. (C) Representative dot plot of eosinophil (CD49b^loSiglec-F⁺) accumulation in the lungs of the indicated mouse strains. Numbers in graph represent percentage of cells in the indicated gates. (D) Percentage (± SEM; n=4) of CD49b^loSiglec-F⁺ cells. (E,F) Quantitative RT-PCR analysis (± SD; n=4) of Angiogenin4 (E) and Mucin2 (F) expression by intestinal epithelial cells isolated from the indicated treatment groups. (G) Alcian blue staining of cryosections of the small intestine. Arrowheads indicate goblet cells with high amounts of mucus production. Scale bar, 100 μm. Original magnification, x20. (H) CCR9 (GFP) expression by intestinal ILC2 and bone marrow ILC2P. Histograms are electronically gated on LSIG cells or ILC2. Open histograms represent analysis of the same population from control mice. (I,J) Representation of GATA3^hi ILCs (i.e., ILC2 or ILC2P) in C57BL/6 (I, upper panel) and Ccr9-deficient (I, lower panel) mice. Numbers represent percentage of cells in the indicated gates. (J) Percentage (± SEM; n=3) of GATA3^hi ILCs in the indicated organs of control (white bars) or Ccr9-deficient (black bars) mice. All data are representative of 3 or more independent experiments. * p ≤ 0.05, ** p ≤ 0.01. LSIG: Lin⁻Sca1^hiId2^hiGATA3^hi bone marrow cells.

Figure 7. GATA3 controls cell fate and maintenance of ILC2

(A) Analyses of RORγt and KLRG1 expression by YFP⁺ (Cre-on) and YFP⁻ (Cre-off) fractions of CD45⁺CD3⁻CD19⁻ lamina propria cells from the indicated mouse strains. Numbers represent percent cells in quadrant. (B) GATA3 expression by intestinal YFP⁺ (Cre-on) RORγt⁺ ILCs of Gata3^+/+ (grey) or Gata3^f/f (open) animals. (C) Expression of GATA3 and KLRG1 by YFP⁺ (Cre-on) and YFP⁻ (Cre-off) fractions of CD45⁺CD3⁻CD19⁻ intestinal lamina propria cells from the indicated mouse strains. Numbers represent percent cells in gate. (D,E) Percentage (± SD; n=4) of KLRG1⁺YFP⁺ cells (D) and of YFP⁺ cells (E) among CD45⁺CD3⁻CD19⁻ lamina propria lymphocytes from the indicated mice. (F) Lamina propria lymphocytes from the indicated mouse strains were stimulated for 4 h with PMA and ionomycin. IL-5 and IL-13 production was analyzed by intracellular cytokine staining and flow cytometry analysis of electronically gated CD45⁺CD3⁻CD19⁻ CD90⁺ lamina propria lymphocytes. Numbers indicate percent cells in quadrant. (G) Highly purified YFP⁻ ILC2 were cultured on mitotically inactivated OP9 feeder cells in the presence of IL-2, IL-7 and 4-OH tamoxifen. After 7 days, cells were analyzed for expression of YFP and KLRG1. Numbers represent percentage of cells in the indicated gates. (H) Expression of Sca1 and YFP among Lin⁻ cells of the bone marrow from the indicated mouse strains. (I) Expression of CD25 and Sca1 by Lin⁻ bone marrow cells. Numbers indicate percentage of cells in quadrant. The lower panel shows the expression of YFP (Cre-on) in electronically gated Lin⁻CD25⁺Sca1^hi bone marrow cells of the indicated mouse strains. Numbers indicate percentage of YFP-positive cells. (J) Highly purified YFP⁺ ILC2P were cultured on mitotically inactivated OP9 feeder cells in the presence of IL-2, IL-7 and 4-OH tamoxifen. After 7 days, the cells were analyzed for expression of YFP and KLRG1. Numbers represent percentage of cells in quadrants. All data are representative of at least two independent experiments. *** p ≤ 0.001, n.s.: not statistically significant. See also Figure S5.