An IFN-γ feedback loop
Innate lymphoid cells (ILCs) play important roles in tissue homeostasis and host defense. Type 1 ILCs (ILC1s) produce interferon-γ (IFN-γ) and require the transcriptional master regulator T-bet. The pathways underlying how these cells develop and differentiate have remained poorly understood. Bai et al. found that the adult mouse liver contains a population of Lin–Sca-1+Mac-1+ hematopoietic stem cells (LSM HSCs) that preferentially differentiate into tissue-resident liver ILC1s. They further show that IFN-γ produced by mature ILC1s promotes the expansion and differentiation of LSM HSCs into ILC1s but not natural killer cells. This work expands our understanding of extramedullary hematopoiesis and underscores the unique immune status of the liver.
Science, this issue p. eaba4177
The predominant sites where hematopoiesis occurs change during the course of mammalian development. Bone marrow (BM) hematopoiesis has long been considered the major source of mature blood cells during adulthood, but extramedullary hematopoiesis in other adult organs can occur under certain circumstances and makes a particularly important contribution when the BM is not functional. In particular, the adult liver environment remains compatible with hematopoiesis and contains a few hematopoietic stem cells (HSCs) with long-term capacity for hematopoietic reconstitution.
The pathways leading to the development of tissue-resident lymphocytes, including liver type 1 innate lymphoid cells (ILC1s), remain unclear. The adult mouse liver ILCs include CD49a−CD49b+ conventional natural killer (cNK) cells and CD49a+CD49b− ILC1s. Given the tissue-resident status of CD49a+CD49b− ILC1s in the liver and their impaired reconstitution in mice receiving BM transplants, we investigated whether liver ILC1s could develop from local hematopoietic progenitors during adulthood.
Previous studies have demonstrated that fetal liver HSCs are enriched in a lineage (Lin)-negative population expressing both Mac-1 and Sca-1. We found that the adult mouse liver also contains Lin−Sca-1+Mac-1+ (LSM) HSCs derived from the fetal liver. An analysis of parabiotic mice showed that adult liver LSM cells were strictly tissue resident at steady state. LSM cells purified from adult mouse liver and transferred into sublethally irradiated immmunodeficient mice by portal vein injection were able to generate multiple hematopoietic lineages but preferentially differentiated into ILC1s rather than cNK cells in the recipient liver. Single-cell RNA sequencing analysis showed that LSM cells represented a complex population of various cell subsets and revealed Lin−CD122+CD49a+ cells as a heterogeneous precursor population downstream from LSM cells, with a differentiation potential restricted to liver ILC1s rather than cNK cells. Mechanistically, we could show that deficiency in the gene encoding interferon-γ (Ifng) or one of its receptors (Ifngr1) selectively reduced the frequency and number of ILC1s and not cNK cells in the liver. Delivery of a plasmid containing the interferon-γ (IFN-γ) cDNA to Ifng-deficient mice via hydrodynamic tail-vein injection selectively increased the frequency and number of liver ILC1s but not of liver cNK cells. Moreover, IFN-γ signaling promoted the expansion and differentiation of LSM cells but not of ILC1s, supporting a model in which IFN-γ acts on these local progenitors to promote liver ILC1 development. Previous studies have shown a strict requirement of the transcription factor T-bet for ILC1 development. We showed that T-bet is not required for LSM cell development but is key for the LSM cell differentiation into ILC1s. We then explored the cellular source of IFN-γ that affects liver ILC1 production. ILC1 numbers were unaffected in the absence of T or B cells. By contrast, Ncr1Cre/+Ifngfl/fl mice, in which Ifng expression is conditionally abolished on NKp46+ cells, harbored a selective deficiency of liver ILC1s. We previously demonstrated that conditional deficiency of the transcription factor Eomes in NKp46+ ILCs leads to an absence of cNK cells, with no impact on liver ILC1s, ruling out a role for cNK cells in liver ILC1 development. Because all NKp46+ ILCs producing IFN-γ are either cNK cells or ILC1s, IFN-γ production by ILC1s therefore promotes the development of ILC1s in the liver through its action on their progenitors.
We identified an IFN-γ–dependent loop that amplifies the development of liver ILC1s but not cNK cells locally. Our findings reveal the contribution of extramedullary hematopoiesis to a distinctive regional immune feature within the liver. These results are reminiscent of the local development of macrophages from embryonic precursors that selectively seed the tissues and of the in situ differentiation of lung ILC2s from tissue-resident progenitors. They advance our knowledge of the importance of extramedullary hematopoiesis to cells of lymphoid origin.
The pathways that lead to the development of tissue-resident lymphocytes, including liver type 1 innate lymphoid cells (ILC1s), remain unclear. We show here that the adult mouse liver contains Lin−Sca-1+Mac-1+ hematopoietic stem cells derived from the fetal liver. This population includes Lin−CD122+CD49a+ progenitors that can generate liver ILC1s but not conventional natural killer cells. Interferon-γ (IFN-γ) production by the liver ILC1s themselves promotes the development of these cells in situ, through effects on their IFN-γR+ liver progenitors. Thus, an IFN-γ–dependent loop drives liver ILC1 development in situ, highlighting the contribution of extramedullary hematopoiesis to regional immune composition within the liver.
Hematopoietic stem cells (HSCs) give rise to multiple lineages of progenitors. The predominant sites where hematopoiesis occurs change during the course of mouse and human development (1, 2). In mice, the first hematopoietic progenitors are found in the yolk sac 7 days post coitus (dpc) and lead to embryonic erythroid cells and myeloid cells (3). The first HSCs initially emerge in the aorta-gonad-mesonephros region of the embryo after 10.5 dpc (4). These cells then colonize the fetal liver at 11 dpc, subsequently expanding and differentiating. Before birth, fetal liver HSCs begin to seed the bone marrow (BM) (3). BM hematopoiesis has long been considered the major source of mature blood cells during adulthood, but extramedullary hematopoiesis in other adult organs, such as the liver and spleen, can occur under certain circumstances and makes a particularly important contribution when the BM is not functional (2). Moreover, several lines of evidence suggest that the adult liver environment remains compatible with hematopoiesis and contains a few HSCs with long-term capacity for hematopoietic reconstitution (5). However, the origin and contribution of adult liver hematopoietic progenitors to local immunological features remain unknown.
The immune composition of the liver differs from that of other organs, with a large number of resident innate immune cells such as type 1 innate lymphoid cells (ILC1s) (6, 7), γδ T cells, and natural killer T (NKT) cells. Adult mouse liver ILCs include CD49a−CD49b+ conventional natural killer (cNK) cells and CD49a+CD49b− ILC1s (8), which can be distinguished on the basis of differences in the expression of CD69, CD200R1, Eomes, T-bet, and TRAIL (fig. S1, A and B). In terms of effector function, liver CD49a+CD49b− ILC1s produce larger amounts of tumor necrosis factor–α, generate smaller quantities of interferon-γ (IFN-γ) and perforin, and have similar levels of granzyme B as compared to CD49a−CD49b+ cNK cells, after stimulation with interleukin-12 (IL-12), IL-15, and IL-18 (fig. S1, C and D). However, the branch point at which the development of liver ILC1s separates from that of liver cNK cells remains unknown. Given the tissue-resident status of CD49a+CD49b− ILC1s in the liver (9) and their impaired reconstitution in mice receiving BM transplants (6), we investigated whether liver ILC1s could develop from local hematopoietic progenitors during adulthood.
Adult mouse liver contains progenitors of liver-resident ILC1s
Previous studies have demonstrated that fetal liver HSCs are enriched in a lineage (Lin)-negative population expressing both Mac-1 and Sca-1 (10). By analyzing the expression of Mac-1 and Sca-1 on CD45+Lin− progenitors from various tissues from wild-type (WT) mice, we found that, like the fetal liver, the adult liver contained Lin−Sca-1+Mac-1+ (LSM) cells, which were present at significantly higher frequencies than in adult BM, peripheral blood, and small intestine lamina propria (siLP) (Fig. 1, A and B, and fig. S2A). Like Lin−Sca-1+c-kit+ (LSK) BM HSCs, some adult liver LSM cells expressed c-kit, but also Flt3 (CD135) and CD34, reminiscent of short-term HSCs (ST-HSCs) and multipotent progenitors, as well as CD93, which is strongly expressed by fetal liver HSCs (11) (Fig. 1C). In addition, unlike long-term HSCs (LT-HSCs) characterized as EPCR+CD150+CD48−(12, 13), most of the adult liver LSM cells were EPCR−CD150−CD48+ (fig. S2B), and only a minor fraction (10 to 20%) expressed the medium-term HSC marker CD49b (14) (fig. S2B). Adult liver LSM cells also strongly expressed the tissue-resident marker CD49a but had low levels of the chemokine receptors CXCR3 and CXCR6 (fig. S2B). Consistent with their heterogeneous cell-surface phenotype, uniform manifold approximation and projection (UMAP) analysis of single-cell RNA sequencing (scRNA-seq) data for sorted LSM cells revealed 12 distinct cell clusters (fig. S3A and table S1). Using previously described hematopoietic progenitor signatures (15), we were able to identify cluster 9 as corresponding to LT-HSCs, cluster 2 as corresponding to ST-HSCs, and cluster 6 as corresponding to common lymphoid progenitors (fig. S3B). Notably, chimera experiments revealed that fetal liver cells reconstituted the pool of liver LSM cells (Fig. 1D) and ILC1s (Fig. 1E) more efficiently than BM cells. In addition, an analysis of parabiotic mice showed that adult liver LSM cells were strictly tissue resident at steady state (fig. S2, C and D). These experiments suggest that LSM cells contain fetal liver–derived multipotent hematopoietic populations, including progenitors that give rise to liver ILC1s. Consistent with this hypothesis, LSM cells purified from adult mouse liver and transferred into sublethally irradiated Rag1−/− mice by portal-vein injection were able to generate multiple hematopoietic lineages (fig. S2E) and preferentially differentiated into CD49a+CD49b− ILC1s rather than cNK cells in the recipient liver (Fig. 1F).