Cutting Edge: Eomesodermin Is Sufficient To Direct Type 1 Innate Lymphocyte Development into the Conventional NK Lineage

Abstract

Type 1 innate lymphocytes comprise two developmentally divergent lineages, type 1 helper innate lymphoid cells (hILC1s) and conventional NK cells (cNKs). All type 1 innate lymphocytes (ILCs) express the transcription factor T-bet, but cNKs additionally express Eomesodermin (Eomes). We show that deletion of Eomes alleles at the onset of type 1 ILC maturation using NKp46-Cre imposes a substantial block in cNK development. Formation of the entire lymphoid and nonlymphoid type 1 ILC compartment appears to require the semiredundant action of both T-bet and Eomes. To determine if Eomes is sufficient to redirect hILC1 development to a cNK fate, we generated transgenic mice that express Eomes when and where T-bet is expressed using Tbx21 locus control to drive expression of Eomes codons. Ectopic Eomes induces cNK-like properties across the lymphoid and nonlymphoid type 1 ILC compartments. Subsequent to their divergent lineage specification, hILC1s and cNKs thus possess substantial developmental plasticity.

Figure 1. Loss of Eomes in NKp46⁺ ILCs impairs cNK development

(A) Representative flow cytometry of CD49a⁺ DX5⁻ hILC1 and CD49a⁻ DX5⁺ cNK subsets among type 1 ILCs [Lin (CD3, Gr-1, TER-119, CD19) ⁻ NK1.1⁺ NKp46⁺] from the indicated organs of wild-type (WT) and Eomes^flox/flox NKp46-Cre⁺ mice (n=4–5 mice per genotype). Absolute numbers of hILC1s and cNKs are indicated. The NK1.1/NKp46 gate is shown at the bottom. (B) Absolute numbers of total type 1 ILCs from the indicated organs of WT, Eomes^flox/flox NKp46-Cre⁺, Tbx21^−/−, and Eomes^flox/flox NKp46-Cre⁺ Tbx21^−/− mice (n=3–5 mice per genotype). Statistics were not calculated (NC) for the difference between WT and Eomes^flox/flox NKp46-Cre⁺ Tbx21^−/− uterus because the Eomes^flox/flox NKp46-Cre⁺ Tbx21^−/− uterus group represents the average of 2 mice. All other data are mean ± SEM representative of 3–5 independent experiments; *p<0.05, **p<0.01, ***p<0.001.

Figure 2. Eomes induces cNK attributes among type 1 ILCs

(A) Transgenic (Tg⁺) Eomes expression in 3 cell populations (from at least 3 mice per genotype) that usually express T-bet but not Eomes: Lin⁻ NK1.1⁺ NKp46⁺ type 1 ILCs from 3-day old neonatal livers; NK1.1⁺ CD3⁺ T cells from adult livers; and CD4⁺ T cells from adult spleens after αCD3- and αCD28-stimulation for 48 hours. Absolute numbers of NK1.1⁺ T cells and CD4⁺ T cells are indicated. Transgenic Eomes does not affect the development of CD4⁺ T cells, but it results in moderate reduction of the number of hepatic NK1.1⁺ T cells. (B) Flow cytometry of CD49a⁺ DX5⁻, CD49a⁺ DX5⁺, and CD49a⁻ DX5⁺ type 1 ILC subsets from the livers and spleens of WT and Tg⁺ mice (n=5 mice per genotype). (C) T-bet and Eomes expression by hepatic CD49a⁺ DX5⁻ (1), CD49a⁺ DX5⁺ (2), and CD49a⁻ DX5⁺ (3) type 1 ILC subsets from WT and Tg⁺ mice (n=4 mice per genotype). (D) Frequency of total type 1 ILCs (among Lin⁻ cells) and frequency of indicated subsets (among total type 1 ILCs) from the livers of WT and Tg⁺ neonates (n=4 mice per genotype). Transgenic Eomes does not affect the overall frequency of neonatal type 1 ILCs. (E) Flow cytometry of TNF-α expression by hepatic type 1 ILCs from WT and Tg⁺ mice (n=4 mice per genotype) after stimulation with 200 ng/mL PMA and 5 μg/mL Ionomycin in the presence of GolgiPlug (Invitrogen) for 4 hours. All data were obtained from mice that have 1 copy of the transgene (Tg⁺). Data are mean ± SEM representative of 2–5 independent analyses; *p<0.05, **p<0.01, ***p<0.001.

Figure 3. Eomes is permissive to some hILC1 attributes

(A) Id2 expression by CD49a⁺ DX5⁻ hILC1s and CD49a⁻ DX5⁺ cNKs among Lin⁻ NK1.1⁺ NKp46⁺ type 1 ILCs from the livers and spleens of Id2^hCD5/+ mice (n=4 mice). (B) Id2 expression by CD49a⁺ Eomes⁻ hILC1s, CD27⁺ Eomes⁺ immature cNKs, and CD27⁻ Eomes⁺ mature cNKs from the spleens of Id2^hCD5/+ mice (n=4 mice). (C) Flow cytometry of Id2 and Eomes expression by NK1.1⁺ NKp46⁺ type 1 ILCs from the livers and spleens of Id2^hCD5/+ and Tg⁺ Id2^hCD5/+ mice (n=5 mice per genotype). Absolute numbers of Id2^hi cells are indicated. (D) Flow cytometry of CD127 and integrin α_v expression by Id2^hi NK1.1⁺ NKp46⁺ type 1 ILCs from the livers and spleens of Id2^hCD5/+ and Tg⁺ Id2^hCD5/+ mice (n=3–4 mice per genotype). (E) Frequency of NKG2A/C/E, Ly49D/G2/H, CD27, CD11b, CD43, KLRG1, and CD122 expression by Id2^hi NK1.1⁺ NKp46⁺ type 1 ILCs from the livers of Id2^hCD5/+ and Tg⁺ Id2^hCD5/+ mice (n=4 mice per genotype). (F) Flow cytometry of helper-like (CD127⁺ integrin α_v+) cells among Ly49⁻ NK1.1⁺ NKp46⁺ type 1 ILCs from the spleens of WT, Tg²⁺, Tbx21^−/−, and Tg²⁺ Tbx21^−/− mice. Absolute numbers of hILC1-like cells in the livers and spleens of n=2 mice per genotype are summarized. (A-F) Data were obtained from mice that have 1 (Tg⁺) or 2 (Tg²⁺) copies of the transgene as indicated. Data are mean ± SEM representative of 2–5 independent experiments; *p<0.05, **p<0.01, ***p<0.001.