Histone methyltransferase Ezh2 negatively regulates NK cell terminal maturation and function.

Natural killer (NK) cells are innate immune cells with the ability to kill tumor cells without prior exposure. NK cells express multiple activating and inhibitory receptors in addition to the low-affinity immunoglobulin G binding receptor CD16. Accumulating evidence implicates a role of NK cells in not only direct killing of tumor cells, but also in cancer immunosurveillance and preventing metastasis to tissues sites. However, at present the distribution, diversity and tissue driven differences in NK cell function are not well characterized which may have an implication on the anti-cancer potential of tissue NK cells. Through collaboration with LiveOnNY our local organ procurement organization, we receive blood, bone marrow (BM), lung, intestines, tonsil and associated lymph nodes (LN) from research consented organ donors. Here we used this unique tissue resource to investigate the distribution, phenotypic, functional and transcriptional diversity of NK cell subsets in human tissues. We found that NK cells are ubiquitously distributed in human tissues comprising up to 40% of the CD45+ CD14/19- cells in blood, BM, spleen and lung, while only a small fraction (up to 2%) in the intestine and the LN. While blood, BM, spleen and lung are dominated by mature NK cells (CD56dim CD16+), majority of the NK cells in intestine and LN are immature (CD56hi CD16-). Age, sex and CMV sero-status do not show any correlation with NK cell distribution or subset frequency in tissues. Notably, NK cell subset distribution seems to drive functional differences between tissue NK cells, with lymphoid site NK cells expressing lower levels of effector molecules granzyme B, TNFα, Prf, Ifnγ and displaying reduced degranulation compared with their counterparts from blood, BM and spleen. For an in-depth analysis of tissue-mediated effects on NK cell subset functionality, we performed whole-transcriptome profiling on immature and mature NK cells isolated from blood, BM, spleen, lung and LN. Our analysis identified several effector molecules and NK cell surface receptors being differentially expressed between immature and mature NK cells. Furthermore, while mature NK cells of blood and tissues have similar transcriptional profiles, the transcriptional profiles of immature blood and tissue NK cells show tissue driven heterogeneity with differential expression of transcription factors, metabolic enzymes and NK cell surface receptors. Interestingly, the transcriptional signature of immature NK cells is reminiscent of the transcriptional signature of tissue resident memory T-cells showing increased expression of CD103, CD49a, CXCR6. We validated the expression of these markers using multiparameter flow cytometry and found that subset of immature NK cells in mucosal sites (lung and intestine) do indeed express markers of tissue residence. Additionally, by applying trajectory projection algorithm on NK cells from tissue sites, we show that resident NK cells comprise a distinct population from immature NK cells. Our study has identified novel, previously unidentified diversity of tissue NK cells. Phenotypic and transcriptional profiling data provide evidence for putative resident NK cells being present in certain tissue sites. Blood NK cells differ from their counterparts in tissues; especially, immature NK cells in tissues may be specifically trained to function in the tissue environment. Finally, due to the uniqueness of phenotypic, functional and transcriptional features of tissue NK cells, they may be more suited to fight cancer in situ in tissues. Citation Format: Pranay Dogra, Takashi Senda, Peter Szabo, Dustin Carpenter, Marta Toth, Puspa Thapa, Mark Snyder, Michelle Miron, Brahma Kumar, Donna L. Farber. Human NK cell distribution memory and residence in tissue sites [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B153.

NK cells are innate-like lymphocytes that eliminate virally infected and cancerous cells, but the mechanisms that control NK cell development and cytotoxicity are incompletely understood. We identified roles for sclerostin domain-containing-1 (Sostdc1) in NK cell development and function. Sostdc1-knockout (Sostdc1 -/-) mice display a progressive accumulation of transitional NK cells (tNKs) (CD27+CD11b+) with age, indicating a partial developmental block. The NK cell Ly49 repertoire in Sostdc1 -/- mice is also changed. Lower frequencies of Sostdc1 -/- splenic tNKs express inhibitory Ly49G2 receptors, but higher frequencies express activating Ly49H and Ly49D receptors. However, the frequencies of Ly49I+, G2+, H+, and D+ populations were universally decreased at the most mature (CD27-CD11b+) stage. We hypothesized that the Ly49 repertoire in Sostdc1 -/- mice would correlate with NK killing ability and observed that Sostdc1-/- NK cells are hyporesponsive against MHC class I-deficient cell targets in vitro and in vivo, despite higher CD107a surface levels and similar IFN-γ expression to controls. Consistent with Sostdc1's known role in Wnt signaling regulation, Tcf7 and Lef1 levels were higher in Sostdc1 -/- NK cells. Expression of the NK development gene Id2 was decreased in Sostdc1-/- immature NK and tNK cells, but Eomes and Tbx21 expression was unaffected. Reciprocal bone marrow transplant experiments showed that Sostdc1 regulates NK cell maturation and expression of Ly49 receptors in a cell-extrinsic fashion from both nonhematopoietic and hematopoietic sources. Taken together, these data support a role for Sostdc1 in the regulation of NK cell maturation and cytotoxicity, and identify potential NK cell niches.

Clonal Tracking of the Geographic Distribution of Hematopoiesis in Nonhuman Primates Provides New Insights into HSPC Migration and Differentiation

Helper innate lymphoid cells (ILCs) combat pathogen infection by rapid secretion of cytokines whereas killer ILCs (NK cells) eliminate virus-infected cells or tumor cells during early defense. However, the early development of NK cells and lineage relationships between NK cells and helper ILCs remain elusive. In PLZF lineage tracing model, normal NK cells are traced at a much lower ratio than helper ILCs, which indicates most NK cells develop from progenitors that do not express PLZF. However, it is also possible that most NK cells develop from PLZF-expressing ILC progenitors (ILCPs), but PLZF-labelled NK cells may proliferate less than unlabeled NK cells. To address these possibilities, we transferred ILCPs and its upstream progenitors early innate lymphoid progenitors (EILPs) and all lymphoid progenitors (ALPs), and then assessed PLZF labelling in NK cells derived from these progenitors. We found that ILCP-derived NK cells were traced at a ratio as high as in helper ILCs whereas EILP and ALP-derived NK cells were labelled at a much lower ratio, which is similar to the ratio in normal NK cells. These results indicate that NK cells develop from both ILCP-dependent and ILCP-independent pathways. Our findings establish novel lineage relationships between NK cells and helper ILCs. We are currently assessing whether different developmental pathways of NK cells lead to functionally distinct subsets of NK cells.

Heterogeneity of Hematopoietic Stem and Progenitor Cell (HSPC) Composition in Αβ T-Cell/CD19 B-Cell Depleted Peripheral Blood Cell Stem Cell (PBSC) Transplant Grafts and Correlation with Immune and Hematopoietic Recovery

The Impact of Aging of Hematopoietic Stem and Progenitor Cells (HSPCs) in Non-Human Primates As Interrogated By Genetic Barcode Clonal Tracking

NK Cell Subsets Isolated From Human Thymus Differ From Peripheral Blood NK Cells in Their Cytotoxic and Cytokine Secreting Capability

Peripheral blood stem cell (PBSC) donation is the primary procedure used to collect hematopoietic stem and progenitor cells (HSPCs) for hematopoietic stem cell transplantation. Single bouts of exercise transiently enrich peripheral blood with HSPCs and cytolytic natural killer cells (CD56dim), which are important in preventing post-transplant complications. To provide a rationale to investigate the utility of exercise in a PBSC donation setting (≈3h), this study aimed to establish whether interval cycling increased peripheral blood HSPC and CD56dim concentrations to a greater degree than continuous cycling. In a randomised crossover study design, eleven males (mean ± SD: age 25 ± 7years) undertook bouts of moderate intensity continuous exercise [MICE, 30min, 65%-70% maximum heart rate (HRmax)], high-volume high intensity interval exercise (HV-HIIE, 4 × 4min, 80%-85% HRmax) and low-volume HIIE (LV-HIIE, 4 × 2min, 90%-95% HRmax). The cumulative impact of each interval on circulating HSPC (CD34+CD45dimSSClow) and CD56dim concentrations (cells/µL), and the bone marrow homing potential of HSPCs (expression of CXCR-4 and VLA-4) were determined. There was an increase in HSPC concentration after two intervals of LV-HIIE (Rest: 1.84 ± 1.55 vs. Interval 2: 2.94 ± 1.34, P = 0.01) and three intervals of HV-HIIE only (Rest: 2.05 ± 0.86 vs. Interval 3: 2.51 ± 1.05, P = 0.04). The concentration of all leukocyte subsets increased after each trial, with this greatest for CD56dim NK cells, and in HIIE vs. MICE (LV-HIIE: 4.77 ± 2.82, HV-HIIE: 4.65 ± 2.06, MICE: 2.44 ± 0.77, P < 0.0001). These patterns were observed for concentration, not frequency of CXCR-4+ and VLA-4+ HSPCs, which was unaltered. There was a marginal decrease in VLA-4, but not CXCR-4 expression on exercise-mobilised HSPCs after all trials (P < 0.0001). The results of the present study indicate that HIIE caused a more marked increase in HSPC and CD56dim NK cell concentrations than MICE, with mobilised HSPCs maintaining their bone marrow homing phenotype. LV-HIIE evoked an increase in HSPC concentration after just 2 × 2-minute intervals. The feasibility and clinical utility of interval cycling in a PBSC donation context should therefore be evaluated.

T-bet and Eomes are T-box transcription factors that drive the differentiation and function of cytotoxic lymphocytes such as NK cells. Their DNA-binding domains are highly similar, suggesting redundant transcriptional activity. However, while these transcription factors have different patterns of expression, the phenotype of loss-of-function mouse models suggests that they play distinct roles in the development of NK cells and other innate lymphoid cells (ILCs). Recent technological advances using reporter mice and conditional knockouts were fundamental in defining the regulation and function of these factors at steady state and during pathological conditions such as various types of cancer or infection. Here, we review these recent developments, focusing on NK cells as prototypical cytotoxic lymphocytes and their development, and also discuss parallels between NK cells and T cells. We also examine the role of T-bet and Eomes in human NK cells and ILC1s. Considering divergent findings on mouse and human ILC1s, we propose that NK cells are defined by coexpression of T-bet and Eomes, while ILC1s express only one of these factors, either T-bet or Eomes, depending on the tissue or the species.

Crosstalk between NFAT and NFκB Signaling Regulates NK Cell Immunosurveillance

Maternal obesity, often linked to the consumption of a high-fat Western-style diet (WSD), poses significant risks to both maternal and fetal health. This review explores the impact of maternal obesity on fetal hematopoietic stem and progenitor cells (HSPCs), highlighting how metabolic and inflammatory shifts in the maternal environment affect HSPC proliferation, differentiation, and long-term immune system development. Maternal obesity leads to hormonal imbalances, increased inflammatory cytokines, placental insufficiency, and altered nutrient availability that disrupt normal HSPC function, potentially predisposing offspring to immune dysfunction, metabolic disorders, and cardiovascular diseases later in life. Notably, maternal obesity skews HSPC differentiation toward the myeloid lineage, which can impair adaptive immune responses and increase the risk of autoimmune diseases and infections. Furthermore, maternal diet-driven epigenetic and transcriptional reprogramming of fetal HSPCs exacerbates chronic inflammation, reinforcing a pro-inflammatory phenotype in downstream progeny that persists into postnatal stages. The review also emphasizes the need for further research to clarify the mechanisms underlying these effects across different species and developmental stages, as well as the potential for targeted interventions to mitigate the adverse impacts of maternal obesity on fetal hematopoiesis and lifelong health outcomes.

NK cells are a major component of the antitumor immune response and are involved in controlling tumor progression and metastases in animal models. Here, we show that dysfunction of these cells accompanies human breast tumor progression. We characterized human peripheral blood NK (p-NK) cells and malignant mammary tumor-infiltrating NK (Ti-NK) cells from patients with noninvasive and invasive breast cancers. NK cells isolated from the peripheral blood of healthy donors and normal breast tissue were used as controls. With disease progression, we found that expression of activating NK cell receptors (such as NKp30, NKG2D, DNAM-1, and CD16) decreased while expression of inhibitory receptors (such as NKG2A) increased and that this correlated with decreased NK cell function, most notably cytotoxicity. Importantly, Ti-NK cells had more pronounced impairment of their cytotoxic potential than p-NK cells. We also identified several stroma-derived factors, including TGF-β1, involved in tumor-induced reduction of normal NK cell function. Our data therefore show that breast tumor progression involves NK cell dysfunction and that breast tumors model their environment to evade NK cell antitumor immunity. This highlights the importance of developing future therapies able to restore NK cell cytotoxicity to limit/prevent tumor escape from antitumor immunity.

The unexpected and differential effect of cyclosporin A on CD56 Bright and CD56 Dim NK cell expansion, phenotype, function and development from progenitor cells

NK Education: Disassociation Between Recovery of Cytoxicity and Cytokine Production In NK Cells After Allogeneic Transplantation.

NK cells are innate immune lymphocytes important for early host defense against infectious pathogens and malignant transformation. MicroRNAs (miRNAs) are small RNA molecules that regulate a wide variety of cellular processes, typically by specific complementary targeting of the 3'UTR of mRNAs. The Dicer1 gene encodes a conserved enzyme essential for miRNA processing, and Dicer1 deficiency leads to a global defect in miRNA biogenesis. In this study, we report a mouse model of lymphocyte-restricted Dicer1 disruption to evaluate the role of Dicer1-dependent miRNAs in the development and function of NK cells. As expected, Dicer1-deficient NK cells had decreased total miRNA content. Furthermore, miRNA-deficient NK cells exhibited reduced survival and impaired maturation defined by cell surface phenotypic markers. However, Dicer1-deficient NK cells exhibited enhanced degranulation and IFN-γ production in vitro in response to cytokines, tumor target cells, and activating NK cell receptor ligation. Moreover, a similar phenotype of increased IFN-γ was evident during acute MCMV infection in vivo. miRs-15a/15b/16 were identified as abundant miRNAs in NK cells that directly target the murine IFN-γ 3'UTR, thereby providing a potential mechanism for enhanced IFN-γ production. These data suggest that the function of miRNAs in NK cell biology is complex, with an important role in NK cell development, survival, or homeostasis, while tempering peripheral NK cell activation. Further study of individual miRNAs in an NK cell specific fashion will provide insight into these complex miRNA regulatory effects in NK cell biology.