Distinct developmental pathways generate functionally distinct populations of natural killer cells.

Natural killer (NK) cells function by eliminating virus-infected or tumor cells. Here we identified an NK-lineage-biased progenitor population, referred to as early NK progenitors (ENKPs), which developed into NK cells independently of common precursors for innate lymphoid cells (ILCPs). ENKP-derived NK cells (ENKP_NK cells) and ILCP-derived NK cells (ILCP_NK cells) were transcriptionally different. We devised combinations of surface markers that identified highly enriched ENKP_NK and ILCP_NK cell populations in wild-type mice. Furthermore, Ly49H+ NK cells that responded to mouse cytomegalovirus infection primarily developed from ENKPs, whereas ILCP_NK cells were better IFNγ producers after infection with Salmonella and herpes simplex virus. Human CD56dim and CD56bright NK cells were transcriptionally similar to ENKP_NK cells and ILCP_NK cells, respectively. Our findings establish the existence of two pathways of NK cell development that generate functionally distinct NK cell subsets in mice and further suggest these pathways may be conserved in humans.

GFI1B specifies developmental potential of innate lymphoid cell progenitors in the lungs.

Tissue-resident innate lymphoid cells (ILCs) play a vital role in the frontline defense of various tissues, including the lung. The development of type 2 ILCs (ILC2s) depends on transcription factors such as GATA3, RORα, GFI1, and Bcl11b; however, the factors regulating lung-resident ILC2s remain unclear. Through fate mapping analysis of the paralog transcription factors GFI1 and GFI1B, we show that GFI1 is consistently expressed during the transition from progenitor to mature ILC2s. In contrast, GFI1B expression is limited to specific subsets of bone marrow progenitors and lung-resident ILC progenitors. We found that GFI1B+ lung ILC progenitors represent a multi-lineage subset with tissue-resident characteristics and the potential to form lung-derived ILC subsets and liver-resident ILC1s. Loss of GFI1B in bone marrow progenitors led to the selective loss of lung-resident IL-18R+ ILCs and mature ILC2, subsequently preventing the emergence of effector ILCs that could protect the lung against inflammatory or tumor challenge.

FOXO1 and FOXO3 Cooperatively Regulate Innate Lymphoid Cell Development.

Natural killer (NK) cells play roles in viral clearance and early surveillance against malignant transformation, yet our knowledge of the underlying mechanisms controlling their development and functions remain incomplete. To reveal cell fate-determining pathways in NK cell progenitors (NKP), we utilized an unbiased approach and generated comprehensive gene expression profiles of NK cell progenitors. We found that the NK cell program was gradually established in the CLP to preNKP and preNKP to rNKP transitions. In line with FOXO1 and FOXO3 being co-expressed through the NK developmental trajectory, the loss of both perturbed the establishment of the NK cell program and caused stalling in both NK cell development and maturation. In addition, we found that the combined loss of FOXO1 and FOXO3 caused specific changes to the composition of the non-cytotoxic innate lymphoid cell (ILC) subsets in bone marrow, spleen, and thymus. By combining transcriptome and chromatin profiling, we revealed that FOXO TFs ensure proper NK cell development at various lineage-commitment stages through orchestrating distinct molecular mechanisms. Combined FOXO1 and FOXO3 deficiency in common and innate lymphoid cell progenitors resulted in reduced expression of genes associated with NK cell development including ETS-1 and their downstream target genes. Lastly, we found that FOXO1 and FOXO3 controlled the survival of committed NK cells via gene regulation of IL-15Rß (CD122) on rNKPs and bone marrow NK cells. Overall, we revealed that FOXO1 and FOXO3 function in a coordinated manner to regulate essential developmental genes at multiple stages during murine NK cell and ILC lineage commitment.

Contribution of Dendritic Cell Subsets to T Cell-Dependent Responses in Mice.

BATF3-deficient mice that lack CD8+ dendritic cells (DCs) showed an exacerbation of chronic graft-versus-host disease (cGVHD), including T follicular helper (Tfh) cell and autoantibody responses, whereas mice carrying the Sle2c2 lupus-suppressive locus with a mutation in the G-CSFR showed an expansion of CD8+ DCs and a poor mobilization of plasmacytoid DCs (pDCs) and responded poorly to cGVHD induction. Here, we investigated the contribution of CD8+ DCs and pDCs to the humoral response to protein immunization, where CD8neg DCs are thought to represent the major inducers. Both BATF3-/- and Sle2c2 mice had reduced humoral and germinal center (GC) responses compared with C57BL/6 (B6) controls. We showed that B6-derived CD4+ DCs are the major early producers of IL-6, followed by CD4-CD8- DCs. Surprisingly, IL-6 production and CD80 expression also increased in CD8+ DCs after immunization, and B6-derived CD8+ DCs rescued Ag-specific adaptive responses in BATF3-/- mice. In addition, inflammatory pDCs (ipDCs) produced more IL-6 than all conventional DCs combined. Interestingly, G-CSFR is highly expressed on pDCs. G-CSF expanded pDC and CD8+ DC numbers and IL-6 production by ipDCs and CD4+ DCs, and it improved the quality of Ab response, increasing the localization of Ag-specific T cells to the GC. Finally, G-CSF activated STAT3 in early G-CSFR+ common lymphoid progenitors of cDCs/pDCs but not in mature cells. In conclusion, we showed a multilayered role of DC subsets in priming Tfh cells in protein immunization, and we unveiled the importance of G-CSFR signaling in the development and function pDCs.

Transition from cMyc to L-Myc during dendritic cell development coordinated by rising levels of IRF8.

During dendritic cell (DC) development, Myc expression in progenitors is replaced by Mycl in mature DCs, but when and how this transition occurs is unknown. We evaluated DC development using reporters for MYC, MYCL, and cell cycle proteins Geminin and CDT1 in wild-type and various mutant mice. For classical type 1 dendritic cells (cDC1s) and plasmacytoid DCs (pDCs), the transition occurred upon their initial specification from common dendritic cell progenitors (CDPs) or common lymphoid progenitors (CLPs), respectively. This transition required high levels of IRF8 and interaction with PU.1, suggesting the use of EICEs within Mycl enhancers. In pDCs, maximal MYCL induction also required the +41kb Irf8 enhancer that controls pDC IRF8 expression. IRF8 also contributed to repression of MYC. While MYC is expressed only in rapidly dividing DC progenitors, MYCL is most highly expressed in DCs that have exited the cell cycle. Thus, IRF8 levels coordinate the Myc-Mycl transition during DC development.

Acute lymphoid leukemia etiopathogenesis.

Acute lymphoid leukemia (ALL) is a type of hematological neoplasm that affects the precursor cells of strains B, T  and NK, with a higher incidence in the pediatric range. The pathophysiology of ALL is characterized by chromosomal abnormalities and genetic alterations involved in the differentiation and proliferation of lymphoid precursor cells. Despite the lack of information in the literature, it is believed that leukemogenesis originates from a complex interaction between environmental and genetic factors, which combined lead to cellular modifications. Environmental factors have been evaluated as possible predisposing factors in the development of ALL but there are still conflicting results in the world literature. In this context, the aim of the present review is to discuss the major exogenous factors regarding ALL.

IL-15 superagonist N-803 improves IFNγ production and killing of leukemia and ovarian cancer cells by CD34+ progenitor-derived NK cells.

Allogeneic natural killer (NK) cell transfer is a potential immunotherapy to eliminate and control cancer. A promising source are CD34 + hematopoietic progenitor cells (HPCs), since large numbers of cytotoxic NK cells can be generated. Effective boosting of NK cell function can be achieved by interleukin (IL)-15. However, its in vivo half-life is short and potent trans-presentation by IL-15 receptor α (IL-15Rα) is absent. Therefore, ImmunityBio developed IL-15 superagonist N-803, which combines IL-15 with an activating mutation, an IL-15Rα sushi domain for trans-presentation, and IgG1-Fc for increased half-life. Here, we investigated whether and how N-803 improves HPC-NK cell functionality in leukemia and ovarian cancer (OC) models in vitro and in vivo in OC-bearing immunodeficient mice. We used flow cytometry-based assays, enzyme-linked immunosorbent assay, microscopy-based serial killing assays, and bioluminescence imaging, for in vitro and in vivo experiments. N-803 increased HPC-NK cell proliferation and interferon (IFN)γ production. On leukemia cells, co-culture with HPC-NK cells and N-803 increased ICAM-1 expression. Furthermore, N-803 improved HPC-NK cell-mediated (serial) leukemia killing. Treating OC spheroids with HPC-NK cells and N-803 increased IFNγ-induced CXCL10 secretion, and target killing after prolonged exposure. In immunodeficient mice bearing human OC, N-803 supported HPC-NK cell persistence in combination with total human immunoglobulins to prevent Fc-mediated HPC-NK cell depletion. Moreover, this combination treatment decreased tumor growth. In conclusion,  N-803 is a promising IL-15-based compound that boosts HPC-NK cell expansion and functionality in vitro and in vivo. Adding N-803 to HPC-NK cell therapy could improve cancer immunotherapy.

In Situ Maturation and Tissue Adaptation of Type 2 Innate Lymphoid Cell Progenitors.

Innate lymphoid cells (ILCs) are generated early during ontogeny and persist predominantly as tissue-resident cells. Here, we examined how ILCs are maintained and renewed within tissues. We generated a single cell atlas of lung ILC2s and found that Il18r1+ ILCs comprise circulating and tissue-resident ILC progenitors (ILCP) and effector-cells with heterogeneous expression of the transcription factors Tcf7 and Zbtb16, and CD103. Our analyses revealed a continuous differentiation trajectory from Il18r1+ ST2- ILCPs to Il18r- ST2+ ILC2s, which was experimentally validated. Upon helminth infection, recruited and BM-derived cells generated the entire spectrum of ILC2s in parabiotic and shield chimeric mice, consistent with their potential role in the renewal of tissue ILC2s. Our findings identify local ILCPs and reveal ILCP in situ differentiation and tissue adaptation as a mechanism of ILC maintenance and phenotypic diversification. Local niches, rather than progenitor origin, or the developmental window during ontogeny, may dominantly imprint ILC phenotypes in adult tissues.

A new lymphoid-primed progenitor marked by Dach1 downregulation identified with single cell multi-omics.

A classical view of blood cell development is that multipotent hematopoietic stem and progenitor cells (HSPCs) become lineage-restricted at defined stages. Lin-c-Kit+Sca-1+Flt3+ cells, termed lymphoid-primed multipotent progenitors (LMPPs), have lost megakaryocyte and erythroid potential but are heterogeneous in their fate. Here, through single-cell RNA sequencing, we identify the expression of Dach1 and associated genes in this fraction as being coexpressed with myeloid/stem genes but inversely correlated with lymphoid genes. Through generation of Dach1-GFP reporter mice, we identify a transcriptionally and functionally unique Dach1-GFP- subpopulation within LMPPs with lymphoid potential with low to negligible classic myeloid potential. We term these 'lymphoid-primed progenitors' (LPPs). These findings define an early definitive branch point of lymphoid development in hematopoiesis and a means for prospective isolation of LPPs.

Protocols for Studying Murine ILC Development.

Understanding the origins and developmental trajectory of innate lymphoid cell (ILC) progenitors has been of substantial interest to the fields of ILC biology and immunology. While mature ILC are rare lymphocytes, ILC progenitors represent an even smaller fraction of cells, providing additional challenges in studying them. Moreover, though the approaches to studying these cells are conceptually straightforward, the technical nuances that underlie them can substantially affect the quality of the data. Herein, we provide a detailed protocol for assessing the frequency of ILC progenitors in the bone marrow, their phenotype, and their potential to develop into mature ILC. These methods make up the foundation of in vivo investigations into ILC development, and we hope these thorough protocols and associated notes facilitate additional, high-quality inquiries into this fascinating field.

Rac2 Regulates the Migration of T Lymphoid Progenitors to the Thymus during Zebrafish Embryogenesis.

The caudal hematopoietic tissue in zebrafish, the equivalent to the fetal liver in mammals, is an intermediate hematopoietic niche for the maintenance and differentiation of hematopoietic stem and progenitor cells before homing to the thymus and kidney marrow. As one of the ultimate hematopoietic organs, the thymus sustains T lymphopoiesis, which is essential for adaptive immune system. However, the mechanism of prethymic T lymphoid progenitors migrating to the thymus remains elusive. In this study, we identify an Rho GTPase Rac2 as a modulator of T lymphoid progenitor homing to the thymus in zebrafish. rac2-Deficient embryos show the inability of T lymphoid progenitors homing to the thymus because of defective cell-autonomous motility. Mechanistically, we demonstrate that Rac2 regulates homing of T lymphoid progenitor through Pak1-mediated AKT pathway. Taken together, our work reveals an important function of Rac2 in directing T lymphoid progenitor migration to the thymus during zebrafish embryogenesis.

Novel, Non-Gene-Destructive Knock-In Reporter Mice Refute the Concept of Monoallelic Gata3 Expression.

Accurately tuned expression levels of the transcription factor GATA-3 are crucial at several stages of T cell and innate lymphoid cell development and differentiation. Moreover, several lines of evidence suggest that Gata3 expression might provide a reliable molecular marker for the identification of elusive progenitor cell subsets at the earliest stages of T lineage commitment. To be able to faithfully monitor Gata3 expression noninvasively at the single-cell level, we have generated a novel strain of knock-in reporter mice, termed GATIR, by inserting an expression cassette encoding a bright fluorescent marker into the 3'-untranslated region of the endogenous Gata3 locus. Importantly, in contrast to three previously published strains of Gata3 reporter mice, GATIR mice preserve physiological Gata3 expression on the targeted allele. In this study, we show that GATIR mice faithfully reflect endogenous Gata3 expression without disturbing the development of GATA-3-dependent lymphoid cell populations. We further show that GATIR mice provide an ideal tool for noninvasive monitoring of Th2 polarization and straightforward identification of innate lymphoid cell 2 progenitor populations. Finally, as our reporter is non-gene-destructive, GATIR mice can be bred to homozygosity, not feasible with previously published strains of Gata3 reporter mice harboring disrupted alleles. The availability of hetero- and homozygous Gata3 reporter mice with an exceptionally bright fluorescent marker, allowed us to visualize allelic Gata3 expression in individual cells simply by flow cytometry. The unambiguous results obtained provide compelling evidence against previously postulated monoallelic Gata3 expression in early T lineage and hematopoietic stem cell subsets.

Hhex regulates murine lymphoid progenitor survival independently of Stat5 and Cdkn2a.

The transcription factor Hhex (hematopoietically expressed homeobox gene) is critical for development of multiple lymphoid lineages beyond the common lymphoid progenitor. In addition, Hhex regulates hematopoietic stem cell (HSC) self-renewal, emergency hematopoiesis, and acute myeloid leukemia initiation and maintenance. Hhex mediates its effects on HSCs and acute myeloid leukemia stem cells via repression of the Cdkn2a tumor suppressor locus. However, we report here that loss of Cdkn2a does not rescue the failure of lymphoid development caused by loss of Hhex. As loss of Hhex causes apoptosis of lymphoid progenitors associated with impaired Bcl2 expression and defective Stat5b signaling, we tested the effects of rescuing these pathways using transgenic mice. Expression of the anti-apoptotic factor Bcl2, but not activated Stat5, rescued the development of T-, B-, and NK-cell lineages in the absence of Hhex. These results indicate that Bcl2 expression, but not Stat5b signaling or loss of Cdkn2a, can overcome the lymphoid deficiencies caused by the absence of Hhex, suggesting that the primary role of this transcription factor is to promote survival of lymphoid progenitors during early lymphoid development.

NK Cell Precursors in Human Bone Marrow in Health and Inflammation.

NK cells are generated from hematopoietic stem cells (HSC) residing in the bone marrow (BM), similar to other blood cells. Development toward mature NK cells occurs largely outside the BM through travel of CD34+ and other progenitor intermediates toward secondary lymphoid organs. The BM harbors multipotent CD34+ common lymphoid progenitors (CLPs) that generate T, B, NK, and Dendritic Cells and are devoid of erythroid, myeloid, and megakaryocytic potential. Over recent years, there has been a quest for single-lineage progenitors predominantly with the objective of manipulation and intervention in mind, which has led to the identification of unipotent NK cell progenitors devoid of other lymphoid lineage potential. Research efforts for the study of lymphopoiesis have almost exclusively concentrated on healthy donor tissues and on repopulation/transplant models. This has led to the widely accepted assumption that lymphopoiesis during disease states reflects the findings of these models. However, compelling evidences in animal models show that inflammation plays a fundamental role in the regulation of HSC maturation and release in the BM niches through several mechanisms including modulation of the CXCL12-CXCR4 expression. Indeed, recent findings during systemic inflammation in patients provide evidence that a so-far overlooked CLP exists in the BM (Lin-CD34+DNAM-1brightCXCR4+) and that it overwhelmingly exits the BM during systemic inflammation. These "inflammatory" precursors have a developmental trajectory toward surprisingly functional NK and T cells as reviewed here and mirror the steady state maintenance of the NK cell pool by CD34+DNAM-1-CXCR4- precursors. Our understanding of NK cell precursor development may benefit from including a distinct "inflammatory" progenitor modeling of lymphoid precursors, allowing rapid deployment of specialized Lin-CD34+DNAM-1brightCXCR4+ -derived resources from the BM.

Cutting Edge: Lymphomyeloid-Primed Progenitor Cell Fates Are Controlled by the Transcription Factor Tal1.

Lymphoid specification is the process by which hematopoietic stem cells (HSCs) and their progeny become restricted to differentiation through the lymphoid lineages. The basic helix-loop-helix transcription factors E2A and Lyl1 form a complex that promotes lymphoid specification. In this study, we demonstrate that Tal1, a Lyl1-related basic helix-loop-helix transcription factor that promotes T acute lymphoblastic leukemia and is required for HSC specification, erythropoiesis, and megakaryopoiesis, is a negative regulator of murine lymphoid specification. We demonstrate that Tal1 limits the expression of multiple E2A target genes in HSCs and controls the balance of myeloid versus T lymphocyte differentiation potential in lymphomyeloid-primed progenitors. Our data provide insight into the mechanisms controlling lymphocyte specification and may reveal a basis for the unique functions of Tal1 and Lyl1 in T acute lymphoblastic leukemia.

Accumulation of Multipotent Hematopoietic Progenitors in Peripheral Lymphoid Organs of Mice Over-expressing Interleukin-7 and Flt3-Ligand.

Interleukin-7 (IL-7) and Flt3-ligand (FL) are two cytokines important for the generation of B cells, as manifested by the impaired B cell development in mice deficient for either cytokine or their respective receptors and by the complete block in B cell differentiation in the absence of both cytokines. IL-7 is an important survival and proliferation factor for B cell progenitors, whereas FL acts on several early developmental stages, prior to B cell commitment. We have generated mice constitutively over-expressing both IL-7 and FL. These double transgenic mice develop splenomegaly and lymphadenopathy characterized by tremendously enlarged lymph nodes even in young animals. Lymphoid, myeloid and dendritic cell numbers are increased compared to mice over-expressing either of the two cytokines alone and the effect on their expansion is synergistic, rather than additive. B cell progenitors, early progenitors with myeloid and lymphoid potential (EPLM), common lymphoid progenitors (CLP) and lineage-, Sca1+, kit+ (LSK) cells are all increased not only in the bone marrow but also in peripheral blood, spleen and even lymph nodes. When transplanted into irradiated wild-type mice, lymph node cells show long-term multilineage reconstitution, further confirming the presence of functional hematopoietic progenitors therein. Our double transgenic mouse model shows that sustained and combined over-expression of IL-7 and FL leads to a massive expansion of most bone marrow hematopoietic progenitors and to their associated presence in peripheral lymphoid organs where they reside and potentially differentiate further, thus leading to the synergistic increase in mature lymphoid and myeloid cell numbers. The present study provides further in vivo evidence for the concerted action of IL-7 and FL on lymphopoiesis and suggests that extramedullary niches, including those in lymph nodes, can support the survival and maintenance of hematopoietic progenitors that under physiological conditions develop exclusively in the bone marrow.

The Majority of CD45- Ter119- CD31- Bone Marrow Cell Fraction Is of Hematopoietic Origin and Contains Erythroid and Lymphoid Progenitors.

The non-hematopoietic cell fraction of the bone marrow (BM) is classically identified as CD45- Ter119- CD31- (herein referred to as triple-negative cells or TNCs). Although TNCs are believed to contain heterogeneous stromal cell populations, they remain poorly defined. Here we showed that the vast majority of TNCs (∼85%) have a hematopoietic rather than mesenchymal origin. Single cell RNA-sequencing revealed erythroid and lymphoid progenitor signatures among CD51- TNCs. Ly6D+ CD44+ CD51- TNCs phenotypically and functionally resembled CD45+ pro-B lymphoid cells, whereas Ly6D- CD44+ CD51- TNCs were enriched in previously unappreciated stromal-dependent erythroid progenitors hierarchically situated between preCFU-E and proerythroblasts. Upon adoptive transfer, CD44+ CD51- TNCs contributed to repopulate the B-lymphoid and erythroid compartments. CD44+ CD51- TNCs also expanded during phenylhydrazine-induced acute hemolysis or in a model of sickle cell anemia. These findings thus uncover physiologically relevant new classes of stromal-associated functional CD45- hematopoietic progenitors.

Impact of FLT3 Receptor (CD135) Detection by Flow Cytometry on Clinical Outcome of Adult Acute Myeloid Leukemia Patients.

BACKGROUND: The significance of FMS-like tyrosine kinase 3 (FLT3)-ITD mutation in acute myeloid leukemia (AML) prognosis has been well established. The aims of this study were to investigate the prognostic impact of the FLT3 protein (CD135) expression and its association with FLT3-ITD mutation, and to identify its role in minimal residual disease. PATIENTS AND METHODS: CD135 was measured by flow cytometry on leukemic blasts of 257 adults with de novo AML. High expression of CD135 ≥ 20% was correlated with clinical, laboratory, and other prognostic factors that influenced treatment outcome. FLT3-ITD mutation was tested by PCR. RESULTS: The frequency of CD135 expression was 138 (53.7%) of 257. FLT3-ITD was detected in (21.4%). Positive CD135 expression was associated with high total leukocyte count (P = .006), platelet count (P = .003), monocytic leukemia (P < .001), and CD34 (P = .008) and CD117 (P = .006) expression. CD135 expression ≥ 25% was a predictor of FLT3-ITD mutation (P = .03). CD135 overexpression was a negative predictor of complete remission and of postinduction minimal residual disease at days 14 and 28 (P < .001). CD135 had an adverse impact on overall and disease-free survival (68.5% vs. 15%, P = .002). Multivariate analysis indicated CD135 was the sole independent prognostic factor for overall survival (hazard ratio = 2.49; 95% confidence interval, 1.855-3.345; P < .001). CONCLUSION: CD135 is emerging as a prognostic factor, a new marker for minimal residual disease, and a potential novel therapeutic target of AML.

NOTCH signaling specifies arterial-type definitive hemogenic endothelium from human pluripotent stem cells.

NOTCH signaling is required for the arterial specification and formation of hematopoietic stem cells (HSCs) and lympho-myeloid progenitors in the embryonic aorta-gonad-mesonephros region and extraembryonic vasculature from a distinct lineage of vascular endothelial cells with hemogenic potential. However, the role of NOTCH signaling in hemogenic endothelium (HE) specification from human pluripotent stem cell (hPSC) has not been studied. Here, using a chemically defined hPSC differentiation system combined with the use of DLL1-Fc and DAPT to manipulate NOTCH, we discover that NOTCH activation in hPSC-derived immature HE progenitors leads to formation of CD144+CD43-CD73-DLL4+Runx1 + 23-GFP+ arterial-type HE, which requires NOTCH signaling to undergo endothelial-to-hematopoietic transition and produce definitive lympho-myeloid and erythroid cells. These findings demonstrate that NOTCH-mediated arterialization of HE is an essential prerequisite for establishing definitive lympho-myeloid program and suggest that exploring molecular pathways that lead to arterial specification may aid in vitro approaches to enhance definitive hematopoiesis from hPSCs.

An ST2-dependent role of bone marrow-derived group 2 innate lymphoid cells in pulmonary fibrosis.

Recent evidence supports that bone marrow (BM)-derived hematopoietic progenitor cells play an important role in lung injury and fibrosis. While these cells give rise to multiple cell types, the ST2 (Il1rl1)-expressing group 2 innate lymphoid cells (ILC2s) derived from BM progenitors have been implicated in tissue repair and remodeling, including in lung fibrosis. To further investigate the precise role of BM-derived ILC2s in the pathogenesis of fibrotic lung disease, their importance in the bleomycin-induced lung fibrosis model was evaluated by analyzing the effects of selective ST2 deficiency in the BM compartment. The results showed that while ST2-sufficient control mice exhibited activation of lung IL-33/ST2 signaling, ILC2 recruitment, IL-13 induction, and fibrosis, these responses were significantly diminished in ST2-deficient-BM chimera mice, with selective loss of ST2 expression only in the BM. This diminished response to bleomycin was similar to that seen in ST2 global knockout mice, suggesting the predominant importance of ST2 from the BM compartment. In wild-type mice, ILC2 recruitment to the lung was accompanied by a concomitant decrease in ST2+ BM cells. ST2-deficient BM cells were unresponsive to IL-33-induced ILC2 maturation. Finally, lineage-negative wild-type, but not ST2-deficient BM cells from bleomycin-treated mice stimulated lung fibroblast type I collagen expression, which was associated with elevated TGFß expression in the BM cells. Taken together, these findings suggested that the BM-derived ILC2s were recruited to fibrotic lung through the IL-33/ST2 pathway, and contributed to fibroblast activation to promote lung fibrosis. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.