Natural Killer Cell Precursors Research Articles

BV and beyond: how to incorporate novel agents into PTCL management.

Peripheral T-cell lymphomas (PTCLs) are a heterogenous yet aggressive group of lymphomas that arise from mature T- or NK-cell precursors. Nodal PTCLs include anaplastic large-cell lymphoma, PTCL not otherwise specified, and follicular helper T-cell lymphomas. Recent advances in understanding these heterogenous diseases have prompted investigation of novel agents to improve on treatment. Brentuximab vedotin, a CD30 antibody-drug conjugate, has been incorporated into frontline treatment regimens of CD30-expressing PTCLs based on the ECHELON-2 trial. Multiple ongoing trials are evaluating the addition of other targeted agents in the frontline and relapsed/refractory setting. These include single-agent brentuximab vedotin, histone deacetylase inhibitors, duvelisib, ruxolitinib, EZH2 inhibitors, and azacitidine, among others. Follicular helper T-cell lymphomas, given frequent mutations in epigenetic regulator genes, may preferentially respond to agents such as histone deacetylase inhibitors, EZH2 inhibitors, and hypomethylating agents. As these therapies evolve in their use for both relapsed/refractory disease and then into frontline treatment, subtype-specific therapy will likely help personalize care for patients with PTCL.

The role of CXCR4-CXCL12 in the development and migration of human natural killer cells

Abstract Natural killer (NK) cells play a critical role in controlling viral infection and malignancy and undergo differentiation from multipotent progenitors in secondary lymphoid tissue. Despite dependence on the microenvironment for differentiation, little is known about the cell-cell interactions that promote NK cell precursor trafficking and maturation in tissue. We have identified the chemokine receptor CXCR4 and its ligand CXCL12 as regulators of the cellular interactions between human NK cell precursors and stromal cells that support NK cell differentiation. Using in-vitro NK cell differentiation, we found that treatment of CD34+ precursors with CXCR4 antagonist AMD3100 leads to a lower efficiency of NK cell generation. Since previous work demonstrates the association of cell migration with NK cell development, we interrogated the effect of AMD3100 on NK cell migration. Using live cell imaging we found that AMD3100 treatment of NK cells or progenitor cells results in altered migratory properties and decreased arrest of cells on CXCL12+ stromal cells. Additional experiments using high resolution microscopy and flow cytometry suggest that integrins are interacting with CXCR4 in NK cells. Our data suggests that the CXCR4-CXCL12 interaction is critical for the migration and proper development of NK cells. Future studies will focus on the necessity for CXCR4-CXCL12 in specific key transitions in development and interrogate the cell biological mechanisms of this process.

PAGER-scFGA: unveiling cell functions and molecular mechanisms in cell trajectories through single-cell functional genomics analysis.

Background: Understanding how cells and tissues respond to stress factors and perturbations during disease processes is crucial for developing effective prevention, diagnosis, and treatment strategies. Single-cell RNA sequencing (scRNA-seq) enables high-resolution identification of cells and exploration of cell heterogeneity, shedding light on cell differentiation/maturation and functional differences. Recent advancements in multimodal sequencing technologies have focused on improving access to cell-specific subgroups for functional genomics analysis. To facilitate the functional annotation of cell groups and characterization of molecular mechanisms underlying cell trajectories, we introduce the Pathways, Annotated Gene Lists, and Gene Signatures Electronic Repository for Single-Cell Functional Genomics Analysis (PAGER-scFGA). Results: We have developed PAGER-scFGA, which integrates cell functional annotations and gene-set enrichment analysis into popular single-cell analysis pipelines such as Scanpy. Using differentially expressed genes (DEGs) from pairwise cell clusters, PAGER-scFGA infers cell functions through the enrichment of potential cell-marker genesets. Moreover, PAGER-scFGA provides pathways, annotated gene lists, and gene signatures (PAGs) enriched in specific cell subsets with tissue compositions and continuous transitions along cell trajectories. Additionally, PAGER-scFGA enables the construction of a gene subcellular map based on DEGs and allows examination of the gene functional compartments (GFCs) underlying cell maturation/differentiation. In a real-world case study of mouse natural killer (mNK) cells, PAGER-scFGA revealed two major stages of natural killer (NK) cells and three trajectories from the precursor stage to NK T-like mature stage within blood, spleen, and bone marrow tissues. As the trajectories progress to later stages, the DEGs exhibit greater divergence and variability. However, the DEGs in different trajectories still interact within a network during NK cell maturation. Notably, PAGER-scFGA unveiled cell cytotoxicity, exocytosis, and the response to interleukin (IL) signaling pathways and associated network models during the progression from precursor NK cells to mature NK cells. Conclusion: PAGER-scFGA enables in-depth exploration of functional insights and presents a comprehensive knowledge map of gene networks and GFCs, which can be utilized for future studies and hypothesis generation. It is expected to become an indispensable tool for inferring cell functions and detecting molecular mechanisms within cell trajectories in single-cell studies. The web app (accessible at https://au-singlecell.streamlit.app/) is publicly available.

NK cell receptor profiling of endometrial and decidual NK cells reveals pregnancy-induced adaptations.

Natural killer (NK) cells, with a unique NK cell receptor phenotype, are abundantly present in the non-pregnant (endometrium) and pregnant (decidua) humanuterine mucosa. It is hypothesized that NK cells in the endometrium areprecursors for decidual NK cells present during pregnancy. Microenvironmental changes can alter the phenotype of NK cells, but it is unclear whether decidual NK cell precursors in the endometrium alter their NK cell receptor repertoire under the influence of pregnancy. To examine whether decidual NK cell precursors reveal phenotypic modifications upon pregnancy, we immunophenotyped the NK cell receptor repertoire of both endometrial and early-pregnancy decidual NK cells using flow cytometry. We showed that NK cells in pre-pregnancy endometrium have a different phenotypic composition compared to NK cells in early-pregnancy decidua. The frequency of killer-immunoglobulin-like receptor (KIR expressing NK cells, especially KIR2DS1, KIR2DL2L3S2, and KIR2DL2S2 was significantly lower in decidua, while the frequency of NK cells expressing activating receptors NKG2D, NKp30, NKp46, and CD244 was significantly higher compared to endometrium. Furthermore, co-expression patterns showed a lower frequency of NK cells co-expressing KIR3DL1S1 and KIR2DL2L3S2 in decidua. Our results provide new insights into the adaptations in NK cell receptor repertoire composition that NK cells in the uterine mucosa undergo upon pregnancy.

The longitudinal characterization of immune responses in COVID-19 patients reveals novel prognostic signatures for disease severity, patients' survival and long COVID.

SARS-CoV-2 pandemic still poses a significant burden on global health and economy, especially for symptoms persisting beyond the acute disease. COVID-19 manifests with various degrees of severity and the identification of early biomarkers capable of stratifying patient based on risk of progression could allow tailored treatments. We longitudinally analyzed 67 patients, classified according to a WHO ordinal scale as having Mild, Moderate, or Severe COVID-19. Peripheral blood samples were prospectively collected at hospital admission and during a 6-month follow-up after discharge. Several subsets and markers of the innate and adaptive immunity were monitored as putative factors associated with COVID-19 symptoms. More than 50 immunological parameters were associated with disease severity. A decision tree including the main clinical, laboratory, and biological variables at admission identified low NK-cell precursors and CD14+CD91+ monocytes, and high CD8+ Effector Memory T cell frequencies as the most robust immunological correlates of COVID-19 severity and reduced survival. Moreover, low regulatory B-cell frequency at one month was associated with the susceptibility to develop long COVID at six months, likely due to their immunomodulatory ability. These results highlight the profound perturbation of the immune response during COVID-19. The evaluation of specific innate and adaptive immune-cell subsets allows to distinguish between different acute and persistent COVID-19 symptoms.

Use of CD122+ natural killer cell precursors for superior anti-leukemia response.

Abstract Acute Myeloid Leukemia (AML) is an aggressive blood cancer in adults. Intensive induction therapy successfully induces a complete response in up to 80% of the adult patients but a fraction of them is refractory or relapses. The fraction of non-responsive or relapsing patients is especially high amongst medically unfit and older patients, who cannot undergo aggressive chemotherapy treatment. Therefore, offering patients failing on first line intensive chemotherapy and medically unfit and older patients an effective treatment option for long-term control or even cure of disease represents a significant yet unmet clinical need. As a component of the innate immunity, NK cells have recently shown great promise for the treatment of patients with different malignancies. Here, we show that injection of human interleukin-2 (IL-2) complexes (IL-2cx) induce the de novo generation and massive expansion of NK cell precursors in vivo. Furthermore, IL-2cx-expanded NK cells exert effector functions with the capacity to control the growth of non-self MHC class I-deficient RMA-S lymphoma cells in vivo, while remaining tolerant towards MHC class-expressing self RMA cells. In an experimental setup mimicking the clinical case of refractory patients after intensive AML induction therapy, IL-2cx treatment mediated strong anti-AML responses following haploidentical bone marrow transplantation without the need for adoptive transfer of donor-derived or in vitro autologous expanded NK cells. Thus, this study demonstrates that IL-2cx immunotherapy allows the in vivo generation and expansion of functionally mature NK cells to achieve long-term response in AML paving the way to an effective treatment option. We are sponsored partly by the Cell Evaluation & Therapy Shared Resource, Hollings Cancer Center, Medical University of South Carolina (P30 CA138313), and an ACS-IRG to CK.

Transcription factor TOX maintains the expression of Mst1 in controlling the early mouse NK cell development.

Rationale: TOX is a DNA-binding factor required for the development of multiple immune cells and the formation of lymph nodes. However, the temporal regulation mode of TOX on NK cell development and function needs to be further explored. Methods: To investigate the role of TOX in NK cells at distinct developmental phases, we deleted TOX at the hematopoietic stem cell stage (Vav-Cre), NK cell precursor (CD122-Cre) stage and late NK cell developmental stage (Ncr1-Cre), respectively. Flow cytometry was used to detect the development and functional changes of NK cell when deletion of TOX. RNA-seq was used to assess the differences in transcriptional expression profile of WT and TOX-deficient NK cells. Published Chip-seq data was exploited to search for the proteins directly interact with TOX in NK cells. Results: The deficiency of TOX at the hematopoietic stem cell stage severely retarded NK cell development. To a less extent, TOX also played an essential role in the physiological process of NKp cells differentiation into mature NK cells. Furthermore, the deletion of TOX at NKp stage severely impaired the immune surveillance function of NK cells, accompanied by down-regulation of IFN-γ and CD107a expression. However, TOX is dispensable for mature NK cell development and function. Mechanistically, by combining RNA-seq data with published TOX ChIP-seq data, we found that the inactivation of TOX at NKp stage directly repressed the expression of Mst1, an important intermediate kinase in Hippo signaling pathway. Mst1 deficient at NKp stage gained the similar phenotype with Toxfl/flCD122Cre mice. Conclusion: In our study, we conclude that TOX coordinates the early mouse NK cell development at NKp stage by maintaining the expression of Mst1. Moreover, we clarify the different dependence of the transcription factor TOX in NK cells biology.

SUBPOPULATION HETEROGENEITY OF NK CELLS DURING THE GENETIC MODIFICATION FOR SUBSEQUENT USE IN TARGETED THERAPY

Obtaining genetically engineered NK cells is one of the developing areas of immunotherapy. In this work, we analyzed the subset heterogeneity of NK cells subjected to retroviral transduction, taking into account the content of adaptive NK cell precursors. It has been shown that subpopulations of KIR2DL2/DL3+, as well as CD57–KIR2DL2/DL3+NKG2C+, can be modified with greater efficiency than the corresponding subpopulations that do not carry the KIR2DL2/DL3 and NKG2C markers. After genetic modification, the CD57–KIR2DL2/DL3+NKG2C+ cells began to express CD57 de novo, acquiring the adaptive NK cell phenotype.

Costimulatory Molecules CD80/86 Trigger Non-Specific Cytotoxic Cell of Nile tilapia (Oreochromis niloticus) to Kill CIK Cells

The teleost non-specific cytotoxic cell (NCC), as the evolutionary precursors of NK cells, is an important cytotoxic cell population in the innate immune system of teleost. We have recently realized that costimulatory CD80/86 have conservation in structural and interactional features with its ligand CD28 in Nile tilapia (Oreochromis niloticus). However, the ability of CD80/86 to regulate NCC activity has not been fully investigated. In the present study, we first obtained the recombinant fusion CD80/86 protein from O. niloticus (rOn-CD80/86). Then, NCC incubation with rOn-CD80/86 resulted in a significant production of NCC effector cytokines, including tumor necrosis factor-alpha, cellular apoptosis susceptibility and NK-lysin. Furthermore, NCC treatment with rOn-CD80/86 could significantly improve the ability to kill kidney cells of Grass carp (CIK) and up-regulate the activities of caspase-1 and caspase-3 in CIKs. The yeast, two-hybrid assay showed that On-CD80/86 cannot directly interact with non-specific cytotoxic cell receptor protein-1 of O. niloticus (On-NCCRP-1). The single-cell RNA-Seq data of Nile tilapia head kidney lymphocytes analysis found On-CD28 did not exhibit expression on NCCs subsets. The above results suggest that costimulatory molecules On-CD80/86 is independent of On-NCCRP-1 and On-CD28 receptor in modulating NCC killing activity in vitro of Nile tilapia. The results also provide more insights into the mechanism of NCC activity regulation.

Cord Blood-Derived Natural Killer Cell Exploitation in Immunotherapy Protocols: More Than a Promise?

Simple SummaryNK cell anti-tumor activity against hematological malignancies is well-established and many studies support their role in the control of solid tumor growth and metastasis generation. However, tumor microenvironment may affect NK cell function. Ongoing studies are aimed to design novel immunotherapeutic protocols to combine NK cell-based immunotherapy with other therapeutic strategies to improve the anti-tumor NK cell response. In this context, UCB is one of the main sources of both mature NK cells and of CD34+ HSPC that can generate NK cells, both in-vivo and in-vitro. UCB-derived NK cells represent a valuable tool to perform in-vitro and preclinical analyses and are already used in several clinical settings, particularly against hematological malignancies. The present review describes the characteristics of different types of UCB-derived NK cells and the in-vitro models to expand them, both for research and clinical purposes in the context of cancer immunotherapy.In the last 20 years, Natural Killer (NK) cell-based immunotherapy has become a promising approach to target various types of cancer. Indeed, NK cells play a pivotal role in the first-line defense against tumors through major histocompatibility complex-independent immunosurveillance. Their role in the control of leukemia relapse has been clearly established and, moreover, the presence of NK cells in the tumor microenvironment (TME) generally correlates with good prognosis. However, it has also been observed that, often, NK cells poorly infiltrate the tumor tissue, and, in TME, their functions may be compromised by immunosuppressive factors that contribute to the failure of anti-cancer immune response. Currently, studies are focused on the design of effective strategies to expand NK cells and enhance their cytotoxic activity, exploiting different cell sources, such as peripheral blood (PB), umbilical cord blood (UCB) and NK cell lines. Among them, UCB represents an important source of mature NK cells and CD34+ Hematopoietic Stem and Progenitor Cells (HSPCs), as precursors of NK cells. In this review, we summarize the UCB-derived NK cell activity in the tumor context, review the different in-vitro models to expand NK cells from UCB, and discuss the importance of their exploitation in anti-tumor immunotherapy protocols.

Sympathetic nervous system-mediated β-adrenergic signaling maintains the pool of mature natural killer cells

Abstract The sympathetic nervous system (SNS) innervates all lymphoid tissues and is, thus, well-positioned to influence the activity of immune cells. Accordingly, the coordinated responses of the SNS and immune system are crucial for immune responses to infection and cancer. Natural killer (NK) cells are essential for the immune response to certain viral infections and cancers in which they kill infected or cancerous cells and produce cytokines and chemokines to instruct the activity of other cells contributing to the response. We find here that steady state SNS signaling maintains the balance of mature and immature NK cells in the spleen and bone marrow. Using 6-hydroxydopamine to ablate sympathetic nerve terminals, we show by single cell RNA-sequencing and flow cytometry that SNS-ablated mice have decreased frequencies of mature CD27− CD11b+ NK cells and increased frequencies of immature CD27+ CD11b− and intermediate CD27+ CD11b+ NK cells. An increased ratio of immature NK cells to mature NK cells has been linked to decreased anti-tumor immunity. Along these lines, SNS-ablation also increases the growth rate of RMA-S tumors, the rejection of which depends on NK cells. This accelerated tumor growth was dependent on the absence of both α- and β-adrenergic signaling. Culture of early NK cell precursors in the presence of selective α- or β-adrenergic agonists suggests that signaling through β-adrenergic receptors promotes the maturation of NK cells. These data suggest that signaling through β-adrenergic receptors mediated by norepinephrine produced by the SNS maintains the pool of mature NK cells, allowing them to effectively survey peripheral tissues and participate in anti-tumor immunity.

The transcription factor Fli1 restricts the formation of memory precursor NK cells during viral infection.

Natural killer (NK) cells are innate lymphocytes that possess traits of adaptive immunity, such as memory formation. However, the molecular mechanisms by which NK cells persist to form memory cells are not well understood. Using single cell RNA sequencing, we identified two distinct effector NK cell (NKeff) populations following mouse cytomegalovirus (MCMV) infection. Ly6C– memory precursor (MP) NK cells displayed enhanced survival during the contraction phase in a Bcl2-dependent manner, and differentiated into Ly6C+ memory NK cells. MP NK cells exhibited distinct transcriptional and epigenetic signatures compared to Ly6C+ NKeff cells, with a core epigenetic signature shared with MP CD8+ T cells enriched in ETS1 and Fli1 DNA-binding motifs. Fli1 was induced by STAT5 signaling ex vivo, and increased levels of the pro-apoptotic factor Bim in early effector NK cells following viral infection. These results suggest that a NK cell-intrinsic checkpoint controlled by the transcription factor Fli1 limits MP NK formation by regulating early effector NK cell fitness during viral infection.

Treatment of patients with acute lymphoblastic leukemia who are not suitable for high-dose chemotherapy and hematopoietic stem cell transplantation

Introduction. Acute lymphoblastic leukemia is a malignant disease characterized by the proliferation of precursor B-cells, Tcells or less often, precursors of NK-cells. B-cell acute lymphoblastic leukemia is more common in patients >60 years of age compared to patients <60 years of age (89% vs. 66%), and cytogenetic abnormalities such as t(9;22) (Ph+) are more common in older than younger patients (36% against 19%). Elderly patients often have a poor status and comorbidities, so poor disease outcome is more common. Clinical and biological features. B cell acute lymphoblastic leukemia is more common in patients >60 years of age compared to patients <60 years of age (89% vs 66%) and cytogenetic abnormalities such as t(9;22) are more common in older vs. younger (36% vs. 19%). Therapy. The elderly and patients with comorbidities require less intensive therapy, based on corticosteroids, vincristine and asparaginase, while avoiding anthracyclines and alkylating agents, due to the high mortality associated with treatment. For ?unfit? patients with Ph-positive acute lymphoblastic leukemia, tyrosine kinase inhibitors with reduced-intensity chemotherapy or corticosteroids alone are recommended. For t(9;22) negative patients, low-dose corticosteroid chemotherapy with or without immunotherapy is recommended. For patients with T-cell acute lymphoblastic leukemia, chemotherapy with venetoclax may be an option. Conclusion. The introduction of targeted therapy has changed treatment options in acute lymphoblastic leukemia. For elderly patients, targeted therapy is a necessary modality since standard chemotherapy leads to a poor outcome due to its toxicity and ineffectiveness.

CD200R1 Distinguishes Uncommitted Precursors from Functionally Mature NK Cells within the Human Tonsil Stage 4A NK Cell Population

Natural killer (NK) cells are cytotoxic innate lymphoid cells (ILCs) whose development and anti-tumor functions can be critical for the successful treatment and long-term disease-free survival of patients with hematologic malignancies. In humans, NK cells derive from bone marrow resident hematopoietic progenitor cells that traffic to secondary lymphoid tissues (SLTs) where they complete their terminal differentiation and maturation through a series of developmental stages before returning to the blood as mature NK cells. Although major stages of human NK cell development in SLTs have been clearly defined according to the differential surface expression of CD34, CD117, CD94, NKp80, CD16, and CD57 among lineage antigen (Lin) negative lymphocytes, continued investigation has revealed additional phenotypic and functional heterogeneity at each developmental stage. Through extensive ex vivo single-cell RNA sequencing and flow cytometry analyses we have identified two subsets of tonsil-resident Lin -CD34 -CD117 +/-CD94 +NKp80 -CD16 -CD57 - stage 4A NK cells. These two subsets differ in their expression of the inhibitory receptor, CD200R1, which is not expressed by mature NK cells in the peripheral blood from healthy individuals. The majority of stage 4A cells expressed high amounts of surface CD200R1, which correlated with low gene and undetectable protein expression of intracellular cytolytic granules (perforin and granzymes A, B, K, and M), killer immunoglobulin-like receptors (KIRs), and transcription factors required for terminal NK cell maturation (EOMES, T-BET). In addition, upon ex vivo stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, CD200R1 + stage 4A NKDIs did not produce interferon-gamma (IFN-g), a hallmark feature of mature NK cells. In contrast, many CD200R1 - stage 4A cells constitutively expressed perforin, granzymes, EOMES, and/or T-BET; many expressed KIRs; and many produced IFN-g upon ex vivo stimulation. Furthermore, the frequency of KIR + cells among CD200R1 - stage 4A cells was significantly higher than that among autologous tonsil stage 4B NK cells (Lin -CD34 -CD117 +/-CD94 +NKp80 +CD16 -CD57 -) (20.8 ± 1.65 vs. 8.12 ± 1.66; p < 0.01; n = 14), suggesting that as a population CD200R1 - stage 4A cells are potentially out of sequence in terms of the linear NK cell developmental pathway. Based on these ex vivo findings, we hypothesized that CD200R1 + stage 4A cells represent NK cell precursors, whereas the CD200R1 - stage 4A population contains more mature NK cells that lack NKp80, CD16, and CD57. To further address this hypothesis and to determine their ex vivo potentials for NK cell and non-NK ILC differentiation, we cultured CD200R1 + and CD200R1 - stage 4A cells in vitro in the presence of OP9-DL1 stroma and recombinant human IL-7 and IL-15, conditions previously shown to support all human ILC and NK cell subset differentiation. Under these conditions, both stage 4A populations generated NKp80 + NK cells in bulk and single-cell clonal assays, whereas neither population gave rise to ILC2s (CD294 +) which precede stage 4A NK cells in the developmental scheme. However, while the majority of cultures derived from CD200R1 + stage 4A clones contained ILC3s (CD94 -NKp44 +), significantly fewer clones from CD200R1 - stage 4A cells produced ILC3s (7 of 26 CD200R1 - clones vs. 20 of 23 CD200R1 + clones; p = 0.000587). Moreover, none of the CD200R1 - stage 4A-derived clonal cultures that contained KIR + NK cells contained ILC3s, suggesting that the majority of CD200R1 - stage 4A cells are lineage committed NK cells. Collectively, these data further characterize the heterogeneity of the human tonsil stage 4A NK cell population and identify CD200R1 as a marker distinguishing uncommitted precursor cells from a minor population of cells with otherwise mature NK-associated phenotype and function. In light of the role of CD200R1 in regulating lymphocyte functions in the setting of cancer, further research is warranted to determine its potential role(s) in regulating human NK cell development. DisclosuresBlachly: KITE: Consultancy, Honoraria; INNATE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria.

EZH2 identifies the precursors of human natural killer cells with trained immunity.

Objective:Trained immunity of natural killer (NK) cells has shown great potential in the treatment of cancers by eliciting enhanced effector responses to restimulation by cytokines or cancer cells for long time periods after preactivation. However, the human NK cells responsible for the generation and maintenance of trained immunity are largely unknown. We hypothesized that heterogeneous human NK cells would respond differentially to stimulation with a combination of IL-12, IL-15, and IL-18, and that an NK cell subset might exist that is mainly responsible for the induction of trained immunity. On the basis of our hypothesis, we aimed to identify the subset from which cytokine-trained human NK cells originate and to explore possible regulatory targets for drug intervention.Methods:Flow cytometry assays were performed to analyze the functions of cytokine-trained NK cells and examine cell division and protein expression in NK cell subsets. Single-cell RNA sequencing (scRNA-seq) plus TotalSeq™ technology was used to track the heterogeneity of NK cells during the induction of trained immunity.Results:Traditional developmental markers for peripheral NK cells were unable to identify the precursors of human NK cells with trained immunity. Therefore, we used scRNA-seq plus TotalSeq™ technology to track the heterogeneity of NK cells during the induction of trained immunity and identified a unique cluster of CD57−NKG2A+EZH2+IFNG+MKI67+IL12R+IL15R+IL18R+ NK cells. Enrichment and pseudotime trajectory analyses suggested that this cluster of NK cells contained the precursor of trained NK cells. We then used flow cytometry to further investigate the role of EZH2 in trained NK precursors and found that CD57−NKG2A+EZH2+ NK cells had faster cell cycles and an enhanced trained phenotype, and EZH2 inhibition significantly impaired the induction of trained immunity in NK cells. These results suggested that EZH2 is a unique epigenetic marker of precursors of human NK cells with trained immunity.Conclusions:Our work revealed human NK heterogeneity in the induction of trained immunity, identified the precursor subset for trained NK cells, and demonstrated the critical role of EZH2 in the induction of trained immunity in human NK cells.

Purinergic Receptor P2Y6 Is a Negative Regulator of NK Cell Maturation and Function.

NK cells are critical innate immune cells that target the tumor cells and cancer-initiating cells and clear viruses by producing cytokines and cytotoxic granules. However, the role of the purinergic receptor P2Y6 in the NK cells remains largely unknown. In this study, we discovered that the expression of P2Y6 was decreased upon the activation of the NK cells. Moreover, in the P2Y6-deficient mice, we found that the deficiency of P2Y6 promoted the development of the NK precursor cells into immature NK and mature NK cells. We also found that the P2Y6 deficiency increased, but the P2Y6 receptor agonist UDP or UDP analog 5-OMe-UDP decreased the production of IFN-γ in the activated NK cells. Furthermore, we demonstrated that the P2Y6-deficient NK cells exhibited stronger cytotoxicity in vitro and antimetastatic effects in vivo. Mechanistically, P2Y6 deletion promoted the expression of T-bet (encoded by Tbx21), with or without the stimulation of IL-15. In the absence of P2Y6, the levels of phospho-serine/threonine kinase and pS6 in the NK cells were significantly increased upon the stimulation of IL-15. Collectively, we demonstrated that the P2Y6 receptor acted as a negative regulator of the NK cell function and inhibited the maturation and antitumor activities of the NK cells. Therefore, inhibition of the P2Y6 receptor increases the antitumor activities of the NK cells, which may aid in the design of innovative strategies to improve NK cell-based cancer therapy.

Malignant NK Cells from EBV(+) Human Extranodal NK/T Cell Lymphoma Exhibit Features of a Distinct NK Cell Developmental Subset Found in Mucosal Associated Lymphoid Tissue

Extranodal natural killer (NK)/T cell lymphoma, nasal type (ENKTL) is an aggressive, rare form of Epstein-Barr virus (EBV)-positive non-Hodgkin lymphoma that typically presents in the naso/oropharynx and is associated with high rates of systemic relapse and poor survival. The neoplastic cells in ENKTL are usually of NK cell origin, yet how they relate to the distinct stages that define normal NK cell development has not been fully described. We have established a model of human NK cell development where early NK cell precursors progress through sequential stages of maturation within mucosal associated lymphoid tissues (MALT) and are terminally differentiated in the peripheral blood (PB) (Freud et al. Cell Reports, 2016). Here we aimed to characterize neoplastic NK cells from ENKTL patients within this NK cell developmental framework.In this study we performed flow cytometry analyses, genome-wide DNA methylation profiling, and RNA sequencing of normal NK cell developmental subsets from 12 healthy donor PB and 12 tonsil specimens and of neoplastic NK cells from PB, bone marrow (BM), or cerebrospinal fluid of 11 patients with relapsed ENKTL. While all 11 of the samples contained NK cells, four of the 11 ENKTL patient samples contained distinctly abnormal NK cell populations that expressed CD56 and CD94 but lacked CD16, CD57, and NKp80. We noted that the recurring CD16(-)CD56(+)CD57(-)CD94(+)NKp80(-) phenotype of these cells was similar to that of MALT-resident “stage 4a” NK cell precursors that are rarely detected in the PB (Figure 1).To further investigate the ENKTL phenotype, we sorted two of the abnormal patient-derived CD16(-)CD56(+)CD57(-)CD94(+)NKp80(-) NK cell populations to >98% purity. Real-time quantitative PCR for the EBNA1 gene confirmed that the sorted cells were EBV(+). We then compared the ENKTL cells to normal NK cell developmental intermediates from healthy donors using genome-wide DNA methylation profiling and global gene expression analysis by RNA sequencing. DNA methylation analyses of human tonsil- and PB-derived NK cell developmental subsets revealed a broad, stage-wise acquisition of an NK cell-specific epigenetic program involving targeted gain and loss of methylation enriched in promoters and enhancer regions of genes with known relevance for NK cell development and function. Hypomethylation during development was found to be highly associated with T-BET, EOMES, ETS, KLF and RUNX family transcription factor binding sites, whereas hypermethylation was associated with RORγt, NFkB, and TCF/E2A (P <1x10-10). Expression of specific transcription factors and NK cell-related genes mirrored observed methylation changes. These analyses also revealed that the DNA methylation patterns of the purified ENKTL cells were most similar to those of MALT-derived stage 4a NK cells (Figure 2).Lastly, we transplanted purified ENKTL cells into immune deficient NOD- scid IL2Rgammanull mice. Whereas normal donor tonsil-derived stage 4a precursor cells underwent terminal differentiation in vivo, the transplanted ENKTL cells maintained the stage 4a-like phenotype and did not show evidence of terminal NK cell differentiation despite extensive expansion and infiltration of numerous organs. Global DNA methylation patterns also remained stable between the primary ENKTL cells and cells collected from the BMs and spleens of engrafted mice. Collectively, the flow cytometry, RNA expression, and epigenetic data show that the CD16(-)CD56(+)CD57(-)CD94(+)NKp80(-) ENKTL phenotype in the patient samples examined is stable and most similar to that of MALT-resident stage 4a NK cell precursors. These findings provide new insight into the biological nature of ENKTL that may guide development of novel therapeutic approaches for this aggressive disease. [Display omitted] DisclosuresBrammer:Celgene: Research Funding. Baiocchi:Prelude therapeutics: Research Funding; essanex: Research Funding; Theravectys: Consultancy; viracta: Membership on an entity's Board of Directors or advisory committees, Research Funding. Porcu:Kura: Research Funding; Miragen: Research Funding; Kiowa: Research Funding; Galderma: Research Funding; Innate Pharma: Research Funding; Tetralogic: Research Funding; Celgene: Research Funding; Cell Medica: Research Funding.

TREM2 promotes natural killer cell development in CD3\u2212CD122+NK1.1+ pNK cells

BackgroundTriggering receptor expressed on myeloid cells 2 (TREM2) signaling is considered to regulate anti-inflammatory responses in macrophages, dendritic cell maturation, osteoclast development, induction of obesity, and Alzheimer’s disease pathogenesis. However, little is known regarding the effect of TREM2 on natural killer (NK) cells.ResultsHere, we demonstrated for the first time that CD3−CD122+NK1.1+ precursor NK (pNK) cells expressed TREM2 and their population increased in TREM2-overexpressing transgenic (TREM2-TG) mice compared with that in female C57BL/6 J wild type (WT) mice. Both NK cell-activating receptors and NK cell-associated genes were expressed at higher levels in various tissues of TREM2-TG mice than in WT mice. In addition, bone marrow-derived hematopoietic stem cells (HSCs) of TREM2-TG mice (TG-HSCs) successfully differentiated into NK cells in vitro, with a higher yield from TG-HSCs than from WT-HSCs. In contrast, TREM2 signaling inhibition by TREM2-Ig or a phosphatidylinositol 3-kinase (PI3K) inhibitor affected the expression of the NK cell receptor repertoire and decreased the expression levels of NK cell-associated genes, resulting in significant impairment of NK cell differentiation. Moreover, in melanoma-bearing WT mice, injection of bone marrow cells from TREM2-TG mice exerted greater antitumor effects than that with cells from WT control mice.ConclusionsCollectively, our data clearly showed that TREM2 promoted NK cell development and tumor regression, suggesting TREM2 as a new candidate for cancer immunotherapy.

HCMV-controlling NKG2C+ NK cells originate from novel circulating inflammatory precursors

There is limited knowledge on the origin and development from CD34+ precursors of the ample spectrum of human natural killer (NK) cells, particularly of specialized NK subsets. This study sought to characterize the NK-cell progeny of CD34+DNAM-1brightCXCR4+ and of other precursors circulating in the peripheral blood of patients with chronic viral infections (eg, HIV, hepatitis C virus, cytomegalovirus reactivation). Highly purified precursors were obtained by flow cytometric sorting and cultured in standard NK-cell differentiation media (ie, SCF, FLT3, IL-7, IL-15). Phenotypic and functional analyses on progenies were performed by multiparametric cytofluorimetric assays. Transcriptional signatures of NK-cell progenies were studied by microarray analysis. Inhibition of cytomegalovirus replication was studied by PCR. Unlike conventional CD34+ precursors, Lin-CD34+DNAM-1brightCXCR4+ precursors from patients with chronic infection, rapidly differentiate into cytotoxic, IFN-γ-secreting CD94/NKG2C+KIR+CD57+ NK-cell progenies. An additional novel subset of common lymphocyte precursors was identified among Lin-CD34-CD56-CD16+ cells and characterized by expression of CXCR4 and lack of perforin and CD94. Lin-CD34-CD56-CD16+Perf-CD94-CXCR4+ precursors are also endowed with generation potential toward memory-like NKG2C+NK cells. Maturing NK-cell progenies mediated strong human cytomegalovirus-inhibiting activity. Microarray analysis confirmed a transcriptional signature compatible with NK-cell progenies and with maturing adaptive NK cells. During viral infections, precursors of adaptive NK cells are released and circulate in the peripheral blood.

The Roles of IL-7 and IL-15 in Niches for Lymphocyte Progenitors and Immune Cells in Lymphoid Organs.

Lymphoid organs consist of immune cells and stromal cells. The stromal cells produce various cytokines that support the development, maintenance, and response of the immune cells. IL-7 and IL-15 are the major cytokines produced by stromal cells and are essential for the development and maintenance of lymphocytes and innate lymphoid cells (ILCs). In addition, IL-7 is indispensable for the organogenesis of lymphoid organs. However, because the amount of these two cytokines is relatively low, it has been difficult to directly detect their expression. Recently, several groups succeeded in establishing IL-7 and IL-15 reporter mouse lines. As expected, IL-7 and IL-15 were detected in mesenchymal stromal cells in the bone marrow and lymph nodes and in epithelial cells in the thymus. Furthermore, IL-7 and IL-15 were differentially expressed in lymphatic endothelial cells and blood endothelial cells, respectively. In addition to their expression, many groups have analyzed the local functions of IL-7 and IL-15 by using cell-type-specific knockout mice. From these experiments, CXCL12-expressing mesenchymal stromal cells were identified as the major niche for early B cell precursors. Single-cell RNA sequencing (scRNA-seq) analysis has revealed different subpopulations of stromal cells in the lymphoid organs, including those that express both IL-7 and IL-15. Future research is still needed to elucidate which stromal cells serve as the niche for the early precursors of ILCs and NK cells in the bone marrow.