TCF-1 upregulation identifies early innate lymphoid progenitors in the bone marrow

Abstract

The cellular and molecular events that drive the early development of innate lymphoid cells (ILCs) remain poorly understood. We show that the transcription factor TCF-1 is required for the efficient generation of all known adult ILC subsets and their precursors. Using novel reporter mice, we identified a new subset of early ILC progenitors (EILPs) expressing high amounts of TCF-1. EILPs lacked efficient T and B lymphocyte potential but efficiently gave rise to NK cells and all known adult helper ILC lineages, indicating that they are the earliest ILC-committed progenitors identified so far. Our results suggest that upregulation of TCF-1 expression denotes the earliest stage of ILC fate specification. The discovery of EILPs provides a basis for deciphering additional signals that specify ILC fate.

Figure 1. TCF-1 is required for the efficient generation of all known adult ILC and their progenitors

(a) Flow cytometry analysis of PLZF⁺ common helper innate lymphoid cell progenitors (CHILP) in the bone marrow of Tcf7^+/+ and Tcf7^−/− littermates. (b) Flow cytometry analysis of NK progenitors (NKP) NK1.1⁻CD122⁺ in pregated Lin⁻ bone marrow cells from Tcf7^+/+ and Tcf7^−/− littermates. (c) Quantification of the numbers of PLZF⁺CHILP (left) and NKP (right) per Tcf7^+/+ or Tcf7^−/− mouse analyzed as in a and b. (d) Flow cytometry analyzing mature ILC, B and T cells in the liver and mesenteric lymph nodes (MLN) of lethally irradiated chimeric mice (CD45.1) intravenously inoculated with Tcf7^−/− or Tcf7^+/+ Lin⁻Sca-1⁺c-Kit⁺ (LSK) bone marrow cells (CD45.2) equally mixed with wild-type competitor LSK cells (CD45.1) and analyzed at 12–16 weeks post-transplant. (e) Donor chimerism of mature ILC, B and T cells in the peripheral tissues of recipient mice as assessed as in d. (f) Flow cytometry analyzing bone marrow CHILP and NKP cells in the chimeric mice at 12–16 weeks post-transplant. (g) Donor chimerism of bone marrow CHILP and NKP as assessed in f. Data are representative of (a, b, d, f) or are pooled from three independent experiments with three mice per group (c, e, g). Error bar = mean ± SEM. *p<0.05. **p<0.01.

Figure 2. Identification of a novel TCF-1-expressing bone marrow cell population, termed early innate lymphoid progenitors (EILP)

(a) Flow cytometry analysis showing the profile of a novel Lin⁻TCF-1⁺IL-7Rα^neg/loThy-1⁻ cell population, termed EILP. Bottom panels depict the levels of surface CD25 and α₄β₇ on EILP. (b) Flow cytometry analyzing the expression of surface CD122, and CXCR6 of the indicated subsets. (c) Expression of the indicated genes in bone marrow common lymphoid progenitors (CLP), EILP, CHILP, and thymus early T lineage progenitors (ETP) and double-negative 3 cells (DN3). Results are normalized to those of the control gene Gapdh. UD, undetectable. Data are representative of more than 10 experiments (a), or are representative of 3 independent experiments (b), or are from triplicate samples representative of 3−6 independent experiments (c). Error bar = mean ± SEM.

Figure 3. Comparison of EILP with bone marrow lymphoid progenitors

(a) Flow cytometry analyzing the expression of Kit, Sca-1 and Flt3 on EILP. LMPP, and CLP. Neg ctrl, negative control. (b) Genome-wide microarray analysis was performed with the indicated progenitor subsets. PCA analysis comparing the transcriptome of EILP with those of other progenitor subsets. (c) Flow cytometry analyzing CD11b⁺ Gr-1⁺ myeloid cells derived from LMPP, CLP and EILP cultured on OP9-stroma with FL and IL-7 for 4 days. (d) Quantification of the number of myeloid cells generated per progenitor input as assessed in c. (e) Flow cytometry analyzing CD11c⁺ DC derived from LMPP, CLP or EILP cultured on OP9-stroma in DC-differentiation cytokines for 4 days. f, Quantification of the number of DC generated per progenitor input as assessed in e. Data are representative of 3 independent experiments (a), or are representative of or from 3 experiments (c, d) or are representative of or from 4 experiments (e,f). Error bar = mean ± SEM. **p<0.01.

Figure 4. EILP efficiently give rise to all four ILC lineages in vivo

(a) Flow cytometry analysis of lymphocytes in the liver, small intestine lamina propria (SiLP), and spleen of Rag2^−/−gc^−/− mice intravenously inoculated with EILP (CD45.2) mixed with bone marrow equivalent numbers of CLP (CD45.1) and analyzed at 3–6 weeks post-transplant. (b) Quantification of the number of donor-derived ILC, B and T cells in the peripheral tissues of the recipient mice as assessed in a. (c) Flow cytometry analyzing the phenotype of liver and SiLP ILC derived from EILP in the Rag2^−/−gc^−/− recipient mice. (d) Flow cytometry analysis of donor-derived DP (double-positive, CD4⁺CD8⁺) thymocytes in sub-lethally irradiated recipient mice (CD45.1) intrathymically inoculated with CLP or EILP (CD45.2) and analyzed at 14 days post-transfer. (e) Quantification of the number of donor-derived DP thymocytes in the recipient mice as assessed in d. Data are representatitve of or from four independent adoptive transfer experiments (a, b, c), or are representative of or from three independent experiments (d,e). Error bar = mean ± SEM. *p<0.05. **p<0.01.

Figure 5. EILP efficiently give rise to all four ILC lienages at the clonal level

(a) Clonogenic differentiation assay showing the emergence of ILC progeny derived from single EILP cultured on OP9 stroma at one-cell per well in the presence of IL-2, IL-7 and SCF for 10 days. Each column represents one well with detected ILC lineages highlighted in grey. Only wells with positive ILC growth are shown. (b) Flow cytometry analyzing the daughter cells derived from EILP single cell culture as assessed in a. (c) Quantification of the number of ILC progeny derived from EILP that were cultured on OP9 or OP9-DL1 in the presence of SCF, IL-2 and IL-7 for 10 days. Data are pooled, or are representative of five independent experiments (b), or are from three independent experiments (c). Error bar = mean ± SEM. *p<0.05. **p<0.01.

Figure 6. EILP persist in the absence of Id2

(a) Flow cytometry analyzing bone marrow EILP from Id2^+/+ Tcf7^GFP and Id2^−/− Tcf7^GFP mice. Non-GFP wildtype mice were used as gating controls. Plots were pre-gated on BM Lin⁻ cells. (b) Quantification of the number of bone marrow CLP, EILP, CHILP and ILC2p from Id2^+/+ Tcf7^GFP and Id2^−/− Tcf7^GFP mice. (c) Expression of the indicated genes by EILP from Id2^+/+ Tcf7^GFP and Id2^−/− Tcf7^GFP mice. Results are normalized to those of the control gene Gapdh. UD, undetectable. Neg ctrl, negative control. Data are representative of or are pooled from three independent experiments with three mice per group (a,b), or are from triplicate samples representative of 2–3 independent experiments (c). Error bar = mean ± SEM. *p<0.05. **p<0.01.