Development Of Lymphoid Cells Research Articles

Decreased PU.1 expression in mature B cells induces lymphomagenesis.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of lymphoma, accounting for 30% of non-Hodgkin lymphomas. Although comprehensive analysis of genetic abnormalities has led to the classification of lymphomas, the exact mechanism of lymphomagenesis remains elusive. The Ets family transcription factor, PU.1, encoded by Spi1, is essential for the development of myeloid and lymphoid cells. Our previous research illustrated the tumor suppressor function of PU.1 in classical Hodgkin lymphoma and myeloma cells. In the current study, we found that patients with DLBCL exhibited notably reduced PU.1 expression in their lymphoma cells, particularly in the non-germinal center B-cell-like (GCB) subtype. This observation suggests that downregulation of PU.1 may be implicated in DLBCL tumor growth. To further assess PU.1's role in mature B cells invivo, we generated conditional Spi1 knockout mice using Cγ1-Cre mice. Remarkably, 13 of the 23 knockout mice (56%) showed splenomegaly, lymphadenopathy, or masses, with some having histologically confirmed B-cell lymphomas. In contrast, no wild-type mice developed B-cell lymphoma. In addition, RNA-seq analysis of lymphoma cells from Cγ1-Cre Spi1F/F mice showed high frequency of each monoclonal CDR3 sequence, indicating that these lymphoma cells were monoclonal tumor cells. When these B lymphoma cells were transplanted into immunodeficient recipient mice, all mice died within 3 weeks. Lentiviral-transduced Spi1 rescued 60% of the recipient mice, suggesting that PU.1 has a tumor suppressor function invivo. Collectively, PU.1 is a tumor suppressor in mature B cells, and decreased PU.1 results in mature B-cell lymphoma development.

Dynamic regulation of innate lymphoid cell development during ontogeny

The helper-like ILC contains various functional subsets, such as ILC1, ILC2, ILC3 and LTi cells, mediating the immune responses against viruses, parasites, and extracellular bacteria, respectively. Among them, LTi cells are also crucial for the formation of peripheral lymphoid tissues, such as lymph nodes. Our research, along with others’, indicates a high proportion of LTi cells in the fetal ILC pool, which significantly decreases after birth. Conversely, the proportion of non-LTi ILCs increases postnatally, corresponding to the need for LTi cells to mediate lymphoid tissue formation during fetal stages and other ILC subsets to combat diverse pathogen infections postnatally. However, the regulatory mechanism for this transition remains unclear. In this study, we observed a preference for fetal ILC progenitors to differentiate into LTi cells, while postnatal bone marrow ILC progenitors preferentially differentiate into non-LTi ILCs. Particularly, this differentiation shift occurs within the first week after birth in mice. Further analysis revealed that adult ILC progenitors exhibit stronger activation of the Notch signaling pathway compared to fetal counterparts, accompanied by elevated Gata3 expression and decreased Rorc expression, leading to a transition from fetal LTi cell-dominant states to adult non-LTi ILC-dominant states. This study suggests that the body can regulate ILC development by modulating the activation level of the Notch signaling pathway, thereby acquiring different ILC subsets to accommodate the varying demands within the body at different developmental stages.

Prospection of Therapeutic Agents Targeting Aurora Kinase, a Protein in the Treatment of Acute Lymphoblastic Leukemia

Background: Acute lymphoblastic leukemia (ALL) is characterized by an imbalance in the production and development of hematopoietic lymphoid cells, a malignant disease capable of affecting the proliferation-selection of hematopoietic cells. The Aurora kinase A protein participates in several steps of the mitosis process. Its deregulation can trigger the process of carcinogenesis, which has become a therapeutic target of interest for computational prediction and the development of inhibitory drugs. Studies report its overexpression in malignant cells of patients with ALL. Objective: The present study aims to prospect new molecules to identify a potential inhibitor of Au-rora kinase A for the pharmaceutical market. Methodology: Virtual screening and molecular docking study was performed using the MCULE and DockThor web servers. The pharmacodynamic and pharmacokinetic profile of the molecules were evaluated using the Swiss ADME and ProTox-II programs were used. Results: Ten molecules were identified by virtual screening, in which only two, MCULE-349 and MCULE-796, showed the best score, binding, at the site of action of the protein, interacting positively with amino acids, lipid-soluble molecules with low toxicity and with violation of only 1 Lipinski rule. Conclusion: Both molecules interact with the site of action, acting as inhibitors or blockers of the catalytic site, becoming potential Aurora kinase A inhibitors and anticancer molecules.

ETS1 Function in Leukemia and Lymphoma.

ETS proto-oncogene 1 (ETS1) is a transcription factor (TF) critically involved in lymphoid cell development and function. ETS1 expression is tightly regulated throughout differentiation and activation in T-cells, natural killer (NK) cells, and B-cells. It has also been described as an oncogene in a range of solid and hematologic cancer types. Among hematologic malignancies, its role has been best studied in T-cell acute lymphoblastic leukemia (T-ALL), adult T-cell leukemia/lymphoma (ATLL), and diffuse large B-cell lymphoma (DLBCL). Aberrant expression of ETS1 in these malignancies is driven primarily by chromosomal amplification and enhancer-driven transcriptional regulation, promoting the ETS1 transcriptional program. ETS1 also facilitates aberrantly expressed or activated transcriptional complexes to drive oncogenic pathways. Collectively, ETS1 functions to regulate cell growth, differentiation, signaling, response to stimuli, and viral interactions in these malignancies. A tumor suppressor role has also been indicated for ETS1 in select lymphoma types, emphasizing the importance of cellular context in ETS1 function. Research is ongoing to further characterize the clinical implications of ETS1 dysregulation in hematologic malignancies, to further resolve binding complexes and transcriptional targets, and to identify effective therapeutic targeting approaches.

IKAROS Family Transcription Factors in Lymphocyte Differentiation and Function.

The IKAROS family of transcription factors comprises four zinc-finger proteins (IKAROS, HELIOS, AIOLOS, and EOS), which over the last decades havebeen established to be critical regulators of the development and function of lymphoid cells. These factors act as homo- or heterodimers and are involved both in gene activation and repression. Their function often involves cross-talk with other regulatory circuits, such as the JAK/STAT, NF-κB, and NOTCH pathways. They control lymphocyte differentiation at multiple stages and are notably critical for lymphoid commitment in multipotent hematopoietic progenitors and for T and Bcell differentiation downstream of pre-TCR and pre-BCR signaling. They also control many aspects of effector functions in mature B and T cells. They are dysregulated or mutated in multiple pathologies affecting the lymphoid system, which range from leukemia to immunodeficiencies. In this chapter, we review the molecular and physiological function of these factors in lymphocytes and their implications in human pathologies.

Genetically corrected RAG2-SCID human hematopoietic stem cells restore V(D)J-recombinase and rescue lymphoid deficiency

AbstractRecombination-activating genes (RAG1 and RAG2) are critical for lymphoid cell development and function by initiating the variable (V), diversity (D), and joining (J) (V(D)J)-recombination process to generate polyclonal lymphocytes with broad antigen specificity. The clinical manifestations of defective RAG1/2 genes range from immune dysregulation to severe combined immunodeficiencies (SCIDs), causing life-threatening infections and death early in life without hematopoietic cell transplantation (HCT). Despite improvements, haploidentical HCT without myeloablative conditioning carries a high risk of graft failure and incomplete immune reconstitution. The RAG complex is only expressed during the G0-G1 phase of the cell cycle in the early stages of T- and B-cell development, underscoring that a direct gene correction might capture the precise temporal expression of the endogenous gene. Here, we report a feasibility study using the CRISPR/Cas9-based “universal gene-correction” approach for the RAG2 locus in human hematopoietic stem/progenitor cells (HSPCs) from healthy donors and RAG2-SCID patient. V(D)J-recombinase activity was restored after gene correction of RAG2-SCID-derived HSPCs, resulting in the development of T-cell receptor (TCR) αβ and γδ CD3+ cells and single-positive CD4+ and CD8+ lymphocytes. TCR repertoire analysis indicated a normal distribution of CDR3 length and preserved usage of the distal TRAV genes. We confirmed the in vivo rescue of B-cell development with normal immunoglobulin M surface expression and a significant decrease in CD56bright natural killer cells. Together, we provide specificity, toxicity, and efficacy data supporting the development of a gene-correction therapy to benefit RAG2-deficient patients.

Lactobacillus-derived indole-3-lactic acid ameliorates colitis in cesarean-born offspring via activation of aryl hydrocarbon receptor

Cesarean section (CS) delivery is known to disrupt the transmission of maternal microbiota to offspring, leading to an increased risk of inflammatory bowel disease (IBD). However, the underlying mechanisms remain poorly characterized. Here, we demonstrate that CS birth renders mice susceptible to dextran sulfate sodium (DSS)-induced colitis and impairs group 3 innate lymphoid cell (ILC3) development. Additionally, CS induces a sustained decrease in Lactobacillus abundance, which subsequently contributes to the colitis progression and ILC3 deficiency. Supplementation with a probiotic strain, L.acidophilus, or its metabolite, indole-3-lactic acid (ILA), can attenuate intestinal inflammation and restore ILC3 frequency and interleukin (IL)-22 level in CS offspring. Mechanistically, we indicate that ILA activates ILC3 through the aryl hydrocarbon receptor (AhR) signaling. Overall, our findings uncover a detrimental role of CS-induced gut dysbiosis in the pathogenesis of colitis and suggest L.acidophilus and ILA as potential targets to re-establish intestinal homeostasis in CS offspring.

Loss of the KN Motif and AnKyrin Repeat Domain 1 (KANK1) Leads to Lymphoid Compartment Dysregulation in Murine Model.

The KN Motif and AnKyrin Repeat Domain 1 (KANK1) is proposed as a tumour suppressor gene, as its expression is reduced or absent in several types of tumour tissue, and over-expressing the protein inhibited the proliferation of tumour cells in solid cancer models. We report a novel germline loss of heterozygosity mutation encompassing the KANK1 gene in a young patient diagnosed with myelodysplastic neoplasm (MDS) with no additional disease-related genomic aberrations. To study the potential role of KANK1 in haematopoiesis, we generated a new transgenic mouse model with a confirmed loss of KANK1 expression. KANK1 knockout mice did not develop any haematological abnormalities; however, the loss of its expression led to alteration in the colony forming and proliferative potential of bone marrow (BM) cells and a decrease in hematopoietic stem and progenitor cells (HSPCs) population frequency. A comprehensive marker expression analysis of lineage cell populations indicated a role for Kank1 in lymphoid cell development, and total protein analysis suggests the involvement of Kank1 in BM cells' cytoskeleton formation and mobility.

The Role of Cytokine-Inducible SH2 Domain-Containing Protein (CISH) in the Regulation of Basal and Cytokine-Mediated Myelopoiesis.

Cytokine-inducible SH2 domain-containing protein (CISH) is a member of the suppressor of cytokine signaling (SOCS) family of negative feedback regulators shown to play crucial roles in lymphoid cell development and function as well as appetite regulation. It has also been implicated in the control of signaling downstream of the receptors for the cytokines granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) in myeloid cells. To investigate the physiological role of CISH in myelopoiesis, mice deficient in CISH were analyzed basally and in response to administration of these cytokines. CISH knockout (KO) mice possessed basally elevated neutrophils in the blood, bone marrow, and spleen compared to wild-type (WT) mice. During GM-CSF-induced myelopoiesis, the frequency of neutrophils, myeloid dendritic cells (DCs), and CFU-M in the bone marrow was higher in the KO, as were the neutrophils and CFU-G in the spleen. In contrast, no differences were observed between KO and WT mice during G-CSF-induced myelopoiesis apart from an elevated frequency of CFU-G and CFU-M in the spleen. This work has identified a role for CISH in the negative regulation of granulopoiesis, including that mediated by GM-CSF.

Cover Image

The cover image is based on the Invited Review Lymphoid cell development from fetal hematopoietic progenitors and human pluripotent stem cells by Shicheng Sun et al., https://doi.org/10.1111/imr.13197

Lymphoid cell development from fetal hematopoietic progenitors and human pluripotent stem cells.

Lymphoid cells encompass the adaptive immune system, including T and B cells and Natural killer T cells (NKT), and innate immune cells (ILCs), including Natural Killer (NK) cells. During adult life, these lineages are thought to derive from the differentiation of long-term hematopoietic stem cells (HSCs) residing in the bone marrow. However, during embryogenesis and fetal development, the ontogeny of lymphoid cells is both complex and multifaceted, with a large body of evidence suggesting that lymphoid lineages arise from progenitor cell populations antedating the emergence of HSCs. Recently, the application of single cell RNA-sequencing technologies and pluripotent stem cell-based developmental models has provided new insights into lymphoid ontogeny during embryogenesis. Indeed, PSC differentiation platforms have enabled de novo generation of lymphoid immune cells independently of HSCs, supporting conclusions drawn from the study of hematopoiesis in vivo. Here, we examine lymphoid development from non-HSC progenitor cells and technological advances in the differentiation of human lymphoid cells from pluripotent stem cells for clinical translation.

Conditional Knock-out of Phf6 Decreases CD44 Expression on Naïve CD4+ T Cells and Skews the Differentiation Toward Regulatory T Cells in Mice

Human PHF6 (plant homeodomain (PHD) finger 6), located in X chromosome, is highly expressed in the central nervous system as well as B- and T-lymphoid cells in human and mice, and involved in multiple physiological pathways through chromatin regulation by interaction with the nucleosome remodeling and deacetylation (NuRD) complex. Germline mutations of PHF6 lead to Börjeson-Forssman-Lehmann syndrome (BFLS), a rare X-linked disorder with distinctive facial features and mental retardation. Meanwhile, somatic PHF6 mutations are present in 11% to 39.5% of adult T-cell acute lymphoblastic leukemia (T-ALL) patients. PHF6 functions as a tumor suppressor gene in T-ALL. Conditional knock-out (cKO) of Phf6 in hematopoietic cells in mice enhances self-renewal potential of hematopoietic stem cells and accelerates the oncogenesis of ICN1 overexpression-induced T-ALL. However, the roles of Phf6 in regulating the development and function of lymphoid cells are not explored yet. In our previous study (YC Hsu et al.Blood adv. 2019), we have shown decreased circulating CD4+ T cells in the Phf6 cKO (Phf6 cKO, Phf6F/F or Phf6F/Y;Vav1-Cre) mice compared with the wild type (WT, Phf6F/F or Phf6F/Y) mice. While the percentages of naïve, effector, and effector memory CD4+ T cells, defined by surface CD44 and CD62L, were comparable between the WT and Phf6 cKO mice, we found significantly lower expression of surface CD44 on naïve CD4+ T cells in the Phf6 cKO mice. To assess the effects of Phf6 on the development of the T cells, we performed bulk RNA sequencing (RNAseq) of thymocytes from 3 pairs of mice. GSEA for these transcriptomes revealed enrichment of the interferon γ, interferon α, and FOXP3 targets. These results support our hypothesis that Phf6 has a role in T cell function and regulation. Consisted with the RNAseq data showing enrichment of the FOXP3 targets in the thymocytes of the Phf6 cKO mice, we discovered that the pooled Treg cells in steady state, containing a majority of natural Treg cells (nTreg cells) and some induced Treg cells (iTreg cells), were significantly increased in the peripheral blood and lymph nodes of the Phf6 cKO mice as revealed by the intracellular Foxp3. (Figure 1a) We further investigated if Phf6 has a role in iTreg cells. To this end, we performed in vitro simulation on splenocytes of the WT and Phf6 cKO mice with CD3 mAB, CD28 mAB, hTGFb1 and mIL2 and found the CD4+Phf6 cKO splenocytes were more prone to differentiate into the CD25+Foxp3+ Treg cells compared with that of the WT mice. (Figure 1b) Collectively, conditional knock-out of Phf6 in mice can skew lymphocyte differentiation toward Treg cells not only in steady state, but also under in vitro regulatory T cell priming condition. To further explore the possible mechanisms, we analyzed single cell RNAseq of splenic T cells of WT and Phf6 cKO mice at steady state and cataloging 7 populations as defined by distinct transcriptomic patterns, including 5 for CD4+ T cells and 2 for CD8+ T cells. (Figure 1c) Compared with the WT, the T cells of Phf6 cKO mice had a larger proportion of Treg cells, which significantly expressed higher Fyb and Eno1. (Figure 1d, e and f) According to the literature, FYB, also known as ADAP (Adhesion and degranulation promoting adaptor protein), is a positive regulator of T-cell development, TCR/CD3-induced T-cell activation, and IL-2 and IFN-g production. (E K Griffiths et al. Science. 2001, Zhang Y et al. Immunology. 2012) Meanwhile, the glycolytic enzyme enolase-1, encoded by Eno1, can stimulate glycolysis, on which the induction and function of induced Treg cells highly depend. (V De Rosa et al. Nat Immunol. 2015) In conclusion, we show that Phf6 is a novel regulator of Treg cells perhaps through ADAP and glycolysis pathways. We will further explore the functions and mechanisms of the Phf6 on T cell regulation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Regulation of myeloid and lymphoid cell development by O-glycans on Notch.

Notch signaling via NOTCH1 stimulated by Delta-like ligand 4 (DLL4) is required for the development of T cells in thymus, and NOTCH2 stimulated by Notch ligand DLL1 is required for the development of marginal zone (MZ) B cells in spleen. Notch signaling also regulates myeloid cell production in bone marrow and is an essential contributor to the generation of early hematopoietic stem cells (HSC). The differentiation program in each of these cellular contexts is optimized by the regulation of Notch signaling strength by O-glycans attached to epidermal growth factor-like (EGF) repeats in the extracellular domain of Notch receptors. There are three major types of O-glycan on NOTCH1 and NOTCH2 - O-fucose, O-glucose and O-GlcNAc. The initiating sugar of each O-glycan is added in the endoplasmic reticulum (ER) by glycosyltransferases POFUT1 (fucose), POGLUT1/2/3 (glucose) or EOGT (GlcNAc), respectively. Additional sugars are added in the Golgi compartment during passage through the secretory pathway to the plasma membrane. Of particular significance for Notch signaling is the addition of GlcNAc to O-fucose on an EGF repeat by the Fringe GlcNAc-transferases LFNG, MFNG or RFNG. Canonical Notch ligands (DLL1, DLL4, JAG1, JAG2) expressed in stromal cells bind to the extracellular domain of Notch receptors expressed in hematopoietic stem cells and myeloid and lymphoid progenitors to activate Notch signaling. Ligand-receptor binding is differentially regulated by the O-glycans on Notch. This review will summarize our understanding of the regulation of Notch signaling in myeloid and lymphoid cell development by specific O-glycans in mice with dysregulated expression of a particular glycosyltransferase and discuss how this may impact immune system development and malignancy in general, and in individuals with a congenital defect in the synthesis of the O-glycans attached to EGF repeats.

TGF-β in patients with hematologic malignancies

TGF-β expression is often increased in diseases such as malignancy, inflammation and fibrotic pathology. TGF-β ligand is involved in many processes of lymphoid cell development. TGF-β exerts an inhibitory stimulus on the differentiation and proliferation of stem cell progenitors. The amount of TGF-β on stem cell progenitors can determine various biological effects. For instance, if high concentrations of TGF-β have an inhibitory effect on stem cell progenitors, at low doses it can become stimulatory, especially in that group of stem cell progenitors that have a high probability of developing into myeloid cells.The aim of the study was to determine the level of TGF-β in the peripheral blood of patients with hematologic malignancies and to see its correlation with blood biomarkers of systemic inflammation.

Development and function of regulatory innate lymphoid cells.

Innate lymphoid cells (ILCs) are a critical element of the innate immune system and are potent producers of pro-inflammatory cytokines. Recently, however, the production of the anti-inflammatory cytokine IL-10 has been observed in all ILC subtypes (ILC1s, ILC2s, and ILC3s) suggesting their ability to adopt a regulatory phenotype that serves to maintain lung and gut homeostasis. Other studies advocate a potential therapeutic role of these IL-10-expressing ILCs in allergic diseases such as asthma, colitis, and pancreatic islet allograft rejection. Herein, we review IL-10 producing ILCs, discussing their development, function, regulation, and immunotherapeutic potential through suppressing harmful inflammatory responses. Furthermore, we address inconsistencies in the literature regarding these regulatory IL-10 producing ILCs, as well as directions for future research.

Delta-like 4-Derived Notch Signals Differentially Regulate Thymic Generation of Skin-Homing CCR10+NK1.1+ Innate Lymphoid Cells at Neonatal and Adult Stages.

The thymus is a primary lymphoid organ for T cell development. Increasing evidence found that the thymus is also an important site for development of innate lymphoid cells (ILCs). ILCs generated in thymi acquire unique homing properties that direct their localization into barrier tissues such as the skin and intestine, where they help local homeostasis. Mechanisms underlying the developmental programming of unique tissue-homing properties of ILCs are poorly understood. We report in this article that thymic stroma-derived Notch signaling is differentially involved in thymic generation of a population of NK1.1+ group 1 ILCs (ILC1s) with the CCR10+ skin-homing property in adult and neonatal mice. We found that thymic generation of CCR10+NK1.1+ ILC1s is increased in T cell-deficient mice at adult, but not neonatal, stages, supporting the notion that a large number of developing T cells interfere with signals required for generation of CCR10+NK1.1+ ILC1s. In an in vitro differentiation assay, increasing Notch signals promotes generation of CCR10+NK1.1+ ILC1s from hematopoietic progenitors. Knockout of the Notch ligand Delta-like 4 in thymic stroma impairs generation of CCR10+NK1.1+ ILC1s in adult thymi, but development of CCR10+NK1.1+ ILC1s in neonatal thymi is less dependent on Delta-like 4-derived Notch signals. Mechanistically, the Notch signaling is required for proper expression of the IL-7R CD127 on thymic NK1.1+ ILC1s, and deficiency of CD127 also impairs thymic generation of CCR10+NK1.1+ ILC1s at adult, but not perinatal, stages. Our findings advanced understanding of regulatory mechanisms of thymic innate lymphocyte development.

Organoids capture tissue-specific innate lymphoid cell development in mice and humans.

Organoid-based models of murine and human innate lymphoid cell precursor (ILCP) maturation are presented. First, murine intestinal and pulmonary organoids are harnessed to demonstrate that the epithelial niche is sufficient to drive tissue-specific maturation of all innate lymphoid cell (ILC) groups in parallel, without requiring subset-specific cytokine supplementation. Then, more complex human induced pluripotent stem cell (hiPSC)-based gut and lung organoid models are used to demonstrate that human epithelial cells recapitulate maturation of ILC from a stringent systemic human ILCP population, but only when the organoid-associated stromal cells are depleted. These systems offer versatile and reductionist models to dissect the impact of environmental and mucosal niche cues on ILC maturation. In the future, these could provide insight into how ILC activity and development might become dysregulated in chronic inflammatory diseases.

ORAI3 is dispensable for store-operated Ca2+ entry and immune responses by lymphocytes and macrophages.

Ca2+ signals regulate the function of many immune cells and promote immune responses to infection, cancer, and autoantigens. Ca2+ influx in immune cells is mediated by store-operated Ca2+ entry (SOCE) that results from the opening of Ca2+ release-activated Ca2+ (CRAC) channels. The CRAC channel is formed by three plasma membrane proteins, ORAI1, ORAI2, and ORAI3. Of these, ORAI1 is the best studied and plays important roles in immune function. By contrast, the physiological role of ORAI3 in immune cells remains elusive. We show here that ORAI3 is expressed in many immune cells including macrophages, B cells, and T cells. To investigate ORAI3 function in immune cells, we generated Orai3-/- mice. The development of lymphoid and myeloid cells in the thymus and bone marrow was normal in Orai3-/- mice, as was the composition of immune cells in secondary lymphoid organs. Deletion of Orai3 did not affect SOCE in B cells and T cells but moderately enhanced SOCE in macrophages. Orai3-deficient macrophages, B cells, and T cells had normal effector functions in vitro. Immune responses in vivo, including humoral immunity (T cell dependent or independent) and antitumor immunity, were normal in Orai3-/- mice. Moreover, Orai3-/- mice showed no differences in susceptibility to septic shock, experimental autoimmune encephalomyelitis, or collagen-induced arthritis. We conclude that despite its expression in myeloid and lymphoid cells, ORAI3 appears to be dispensable or redundant for physiological and pathological immune responses mediated by these cells.

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-15Rβ (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.

In vivo impact of JAK3 A573V mutation revealed using zebrafish

BackgroundJanus kinase 3 (JAK3) acts downstream of the interleukin-2 (IL-2) receptor family to play a pivotal role in the regulation of lymphoid cell development. Activating JAK3 mutations are associated with a number of lymphoid and other malignancies, with mutations within the regulatory pseudokinase domain common.MethodsThe pseudokinase domain mutations A572V and A573V were separately introduced into the highly conserved zebrafish Jak3 and transiently expressed in cell lines and zebrafish embryos to examine their activity and impact on early T cells. Genome editing was subsequently used to introduce the A573V mutation into the zebrafish genome to study the effects of JAK3 activation on lymphoid cells in a physiologically relevant context throughout the life-course.ResultsZebrafish Jak3 A573V produced the strongest activation of downstream STAT5 in vitro and elicited a significant increase in T cells in zebrafish embryos. Zebrafish carrying just a single copy of the Jak3 A573V allele displayed elevated embryonic T cells, which continued into adulthood. Hematopoietic precursors and NK cells were also increased, but not B cells. The lymphoproliferative effects of Jak3 A573V in embryos was shown to be dependent on zebrafish IL-2Rγc, JAK1 and STAT5B equivalents, and could be suppressed with the JAK3 inhibitor Tofacitinib.ConclusionsThis study demonstrates that a single JAK3 A573V allele expressed from the endogenous locus was able to enhance lymphopoiesis throughout the life-course, which was mediated via an IL-2Rγc/JAK1/JAK3/STAT5 signaling pathway and was sensitive to Tofacitinib. This extends our understanding of oncogenic JAK3 mutations and creates a novel model to underpin further translational investigations.