Natural Killer Cells In Bone Marrow Research Articles

Myc Regulates the Development and Anti-Tumor Immunity of Natural Killer Cells

Natural Killer (NK) cells play an essential role in the innate immunity. We recently reported that in aggressive NK cell leukemia, MYC is hyperactivated and responsible for the survival and proliferation of leukemic cells through the activation of JAK/STAT pathway (Huang et al., Cell Research 2017). However, the role of Myc in normal NK cell development or function has not been fully explored. Here we addressed the important question on whether Myc is required for NK cell development and its anti-tumor immunity.By crossing Mycf/f allele with Ncr1Cre allele, we generated a NK cell specific Myc conditional knock out (cKO) mouse model. Mycf/f mice were used as control. The percentages of NK1.1+DX5+ mature NK cells (mNK) decreased significantly in both the peripheral blood (PB) and spleen compared to the control mice. In addition, the percentages of terminally differentiated CD11b+CD27- cells within the mNK population decreased, while CD11b-CD27+ cells increased. The expression of terminal differentiation marker Klrg1 also significantly decreased. Further analyzing the NK cells in the bone marrow (BM) revealed the CD122+% and mNK% in the BM did not change. In contrast, the NK1.1-DX5-% in lin-CD122+ population (NK progenitors) increased by 1-fold. This suggested that the development of NK cells was impaired after Myc deletion.To investigate the effects of overexpressed Myc on normal NK cell development, we generated a MYC inducible mouse model (iMYC) by crossing Tg(tetO-MYC) allele with Ncr1Cre and Rosa26Loxp-Stop-Loxp-rtTA alleles. Mice without iMYC allele were used as controls. We transplanted the BM cells from the control or iMYC mice into lethally irradiated BoyJ recipients. We observed that mNK% in CD45.2+ cells increased significantly in liver and PB 3 weeks after the transplantation and dox induction. The ratio of CD27-:CD27+ cells in NK1.1+DX5+CD11b+ cells and Klrg1+mNK% also increased in all tissues analyzed (BM, liver, PB, spleen). In addition, Brdu incorporation rates were increased in spleen and BM NK cells from the iMYC mice. NK cells isolated from iMYC mice showed an enhanced proliferation ability in vitro by dox induction in a dose-dependent manner. These suggested MYC promoted the NK cell commitment, maturation and proliferation.To explore whether the NK cell cytotoxic functions were also affected by Myc, we measured the IFN-γ secretion and CD107a degranulation by PMA/ionomycin or YAC-1 stimulation. Both of the abilities were significantly impaired in Myc cKO mice. Furthermore, the direct lysis of YAC-1 cells by NK cells was also decreased. In contrast, the CD107a degranulation did not change in iMYC mice. The secretion of IFN-γ of splenic NK cells after PMA/Ionomycin stimulation was slightly decreased in iMYC mice, which may be due to the increased population of terminally differentiated NK cells with lower cytotoxicity. This suggested that the physiological level of Myc was important for the cytotoxic functions of NK cells.To investigate whether Myc deletion in NK cells affects the tumor surveillance, we used 2 syngeneic tumor models B16F10 (melanoma) and MC38 (colon adenocarcinoma). The lung metastatic dots were significantly increased in Myc cKO mice compared to control 2 weeks after the inoculation of B16F10. All of the mice in the Myc cKO group were dead on day 21 while the survival rates were 100% in the control group. The weights and volumes of tumors in mice subcutaneously inoculated with MC38 were also significantly higher in Myc cKO mice. This indicated a key role of Myc in NK cell mediated immunity.Finally, we performed transcriptome analysis of splenic NK cells sorted from Myc cKO mice and control mice. The top 2 enriched KEGG pathways in the down-regulated genes includes NK cell mediated cytotoxicity. Interestingly, the expression of Cish, which is an important NK cell anti-tumor immunity checkpoint, was significantly increased in the Myc cKO group, suggesting Myc may affect the NK cell functions through the downregulation of Cish.In summary, our data showed that Myc promoted NK cell development and proliferation. A physiological level of Myc was critical for NK cell cytotoxicity and anti-tumor immunity. Our data also indicated that targeting Myc could be a potential strategy in boosting NK cell-mediated immunotherapy or NK cell expansion ex vivo. Further studies are needed to uncover the underlying mechanism and explore the translational application. DisclosuresNo relevant conflicts of interest to declare.

Fratricide of NK Cells in Daratumumab Therapy for Multiple Myeloma Overcome by Ex Vivo-Expanded Autologous NK Cells.

Purpose: Daratumumab and its use in combination with other agents is becoming a new standard of care for the treatment of multiple myeloma. We mechanistically studied how daratumumab acts on natural killer (NK) cells.Experimental Design: Quantities of NK cells in peripheral blood and/or bone marrow of patients with multiple myeloma or healthy donors were examined by flow cytometry. NK-cell apoptosis and the associated mechanism were assessed by flow cytometry and immunoblotting. Patients' NK cells were expanded in vitro using feeder cells. Combination treatment of daratumumab and expanded NK cells was performed using an MM.1S xenograft animal model.Results: CD38-/low NK cells survived, whereas CD38+ NK cells were almost completely eliminated, in peripheral blood and bone marrow of daratumumab-treated multiple myeloma patients. NK-cell depletion occurred due to daratumumab-induced NK-cell fratricide via antibody-dependent cellular cytotoxicity. Consequently, CD38-/low NK cells were more effective for eradicating multiple myeloma cells than were CD38+ NK cells in the presence of daratumumab. Blockade of CD38 with the F(ab)2 fragments of daratumumab inhibited the antibody-mediated NK-cell fratricide. CD38-/low NK cells displayed a significantly better potential for expansion than CD38+ NK cells, and the expanded NK cells derived from the former population were more cytotoxic than those derived from the latter against multiple myeloma cells. Therefore, infusion of ex vivo-expanded autologous NK cells from daratumumab-treated patients may improve the antibody therapy.Conclusions: We unravel a fratricide mechanism for daratumumab-mediated NK-cell depletion and provide a potential therapeutic strategy to overcome this side effect in daratumumab-treated patients with multiple myeloma. Clin Cancer Res; 24(16); 4006-17. ©2018 AACR.

A novel mechanism of innate immune dysfunction in untreated Chronic Lymphocytic Leukemia.

Abstract The consequences of immune dysfunction in B-Chronic Lymphocytic Leukemia (CLL) likely relate to the incidence of serious recurrent infections and second malignancies that plague CLL patients. The known immune abnormalities are not able to consistently explain these complications and it is not easy to correct their immune status. Here, we report bone marrow (BM) hematopoietic dysfunction in early and late stage untreated CLL patients. We found reduced functional capacity of hematopoietic progenitors in BM using colony forming unit assays and flow cytometry revealed significant reductions in frequencies of hematopoietic stem and progenitor cell (HSPC) populations. Consistent with the reduction in HSPCs, monocytes and natural killer cells in BM were reduced and the reduction in BM was recapitulated in blood. Finally, we report imbalances in protein levels of the transcriptional regulators HIF-1α, PU.1, and GATA-2 in CLL patient BM, providing molecular insight into the basis of HSPC dysfunction. Importantly, PU.1 and GATA-2 were rapidly increased when healthy HSPCs were exposed in vitro to TNFα, a cytokine constitutively produced by CLL B cells. Together, these findings reveal BM hematopoietic dysfunction in untreated CLL patients, adding to the complex immunodeficiency state of these patients and the reduced capacity of the innate immune system.

Effect of the invivo application of granulocyte colony-stimulating factor on NK cells in bone marrow and peripheral blood.

Granulocyte colony‐stimulating factor (G‐CSF) has been widely used in the field of allogeneic haematopoietic stem cell transplantation (allo‐HSCT) for priming donor stem cells from the bone marrow (BM) to peripheral blood (PB) to collect stem cells more conveniently. Donor‐derived natural killer (NK) cells have important antitumour functions and immune regulatory roles post‐allo‐HSCT. The aim of this study was to evaluate the effect of G‐CSF on donors' NK cells in BM and PB. The percentage of NK cells among nuclear cells and lymphocyte was significantly decreased and led to increased ratio of T and NK cells in BM and PB post‐G‐CSF in vivo application. Relative expansion of CD56bri NK cells led to a decreased ratio of CD56dim and CD56bri NK subsets in BM and PB post‐G‐CSF in vivo application. The expression of CD62L, CD54, CD94, NKP30 and CXCR4 on NK cells was significantly increased in PB after G‐CSF treatment. G‐CSF treatment decreased the IFN‐γ‐secreting NK population (NK1) dramatically in BM and PB, but increased the IL‐13‐secreting NK (NK2), TGF‐β‐secreting NK (NK3) and IL‐10‐secreting NK (NKr) populations significantly in BM. Clinical data demonstrated that higher doses of NK1 infused into the allograft correlated with an increased incidence of chronic graft‐vs‐host disease post‐transplantation. Taken together, our results show that the in vivo application of G‐CSF can modulate NK subpopulations, leading to an increased ratio of T and NK cells and decreased ratio of CD56dim and CD56bri NK cells as well as decreased NK1 populations in both PB and BM.

206 - Clinical Significance of Determination of Bone Marrow Natural Killer Cells (NK); Soluble TNF-a and IL-32 Levels in MDS Patients

206 - Clinical Significance of Determination of Bone Marrow Natural Killer Cells (NK); Soluble TNF-a and IL-32 Levels in MDS Patients

NAP-2 Secreted by Human NK Cells Can Stimulate Mesenchymal Stem/Stromal Cell Recruitment.

SummaryStrategies for improved homing of mesenchymal stem cells (MSCs) to a place of injury are being sought and it has been shown that natural killer (NK) cells can stimulate MSC recruitment. Here, we studied the chemokines behind this recruitment. Assays were performed with bone marrow human MSCs and NK cells freshly isolated from healthy donor buffy coats. Supernatants from MSC-NK cell co-cultures can induce MSC recruitment but not to the same extent as when NK cells are present. Antibody arrays and ELISA assays confirmed that NK cells secrete RANTES (CCL5) and revealed that human NK cells secrete NAP-2 (CXCL7), a chemokine that can induce MSC migration. Inhibition with specific antagonists of CXCR2, a receptor that recognizes NAP-2, abolished NK cell-mediated MSC recruitment. This capacity of NK cells to produce chemokines that stimulate MSC recruitment points toward a role for this immune cell population in regulating tissue repair/regeneration.

CD69 and CXCR6 Identify a Distinct Population of Human Bone Marrow and Spleen Resident NK Cells

In recent years, evidence has been provided that natural killer (NK) cells can function as anti-leukemic effector cells. In humans, two NK cell populations are usually distinguished: CD56dimCD16+ NK cells form the predominant population in blood while the CD56brightCD16- NK cell population is more prominent in tissues. However, little data regarding tissue specific characteristics of human NK cells are available, especially in bone marrow as an important localization of leukemic cells. Therefore, we evaluated the expression of chemokine receptors and adhesion molecules on NK cells in healthy donor blood, bone marrow and spleen by flow cytometry.Besides the two conventional NK cell subsets, a major third NK cell population was identified in bone marrow and spleen based on the combined expression of CD69 and CXCR6 (Figure 1). CD69+CXCR6+ NK cells represented 9-50% (mean 30%) of NK cells in marrow (n=15) and 26-57% (mean 43%) of NK cells in spleen (n=7). This CD69+CXCR6+ population was not detected in blood (n=15) nor in cord blood, neither mobilized into the blood stream after G-CSF treatment. The ratio and phenotype of the remaining conventional CD56bright and CD56dim NK cells in bone marrow and spleen were comparable to blood. Early after pediatric hematopoietic stem cell transplantation, CD69+CXCR6+ NK cells were absent in bone marrow, but gradually reached normal levels within the first year after transplantation.CD56 was expressed on marrow and spleen CD69+CXCR6+ NK cells at slightly lower levels than the conventional CD56bright NK cells and CD16 was expressed by 7-30% of CD69+CXCR6+ NK cells. CD69+CXCR6+ NK cells expressed high levels of the adhesion molecule CD54 (ICAM-1) as well as natural cytotoxicity triggering receptor NKp46 compared to the conventional CD56bright and CD56dim NK cell populations. CD69+CXCR6+ NK cells did not express the early differentiation markers CD117 (c-kit) and CD127 (IL7Rα), which are expressed by immature NK cells, type III innate lymphoid cells and, to some extent, by conventional CD56bright NK cells. The inhibitory receptor NKG2A, which is acquired early in maturation but lost during the differentiation of CD56dim NK cells, was expressed by 60% of CD69+CXCR6+ NK cells. Furthermore, CD69+CXCR6+ NK cells did not express markers acquired late in differentiation (KIRs, CD57, KLRG1, NKG2C).In functional experiments assessing cytokine producing capacity, CD69+CXCR6+ NK cells were comparable to the CD56dim subset, requiring the combined stimulation with IL12+IL15+IL18 to produce IFN-γ. However, with respect to cytotoxic potential, CD69+CXCR6+ NK cells more resembled the CD56bright NK cell subset; in resting state, CD69+CXCR6+ NK cells expressed perforin but not granzyme B, which was upregulated during overnight IL12+IL15 stimulation. Upon co-culture with K562 tumor cells, CD69+CXCR6+ NK cells degranulated (CD107a) at levels comparable to the conventional CD56bright and CD56dim NK cells.In summary, we identified a distinct NK cell population in human bone marrow and spleen. These cells were a) absent in blood, b) expressed tissue retention marker CD69, and c) were present alongside the conventional NK cell subsets in marrow and spleen. Together, these findings indicate that NK cells with the discriminative CD69+CXCR6+ phenotype constitute a tissue resident NK cell subset. Furthermore, CD69+CXCR6+ NK cells did not express CD49a+, distinguishing them from the recently described liver resident NK cells (Marquardt et al, 2015). Based on their surface receptor expression profile and functional characteristics, CD69+CXCR6+ NK cells have a mature signature that differs from both conventional NK cell subsets. Additional studies in healthy individuals and patients with immuno-hematological diseases are needed to investigate the immunological function of CD69+CXCR6+ NK cells. For example, these cells could have a local immunoregulatory or effector function. Alternatively, bone marrow and spleen may form a reservoir for effector cells waiting to be released into the blood stream. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bone-Marrow-Resident NK Cells Prime Monocytes for Regulatory Function during Infection

Tissue-infiltrating Ly6C(hi) monocytes play diverse roles in immunity, ranging from pathogen killing to immune regulation. How and where this diversity of function is imposed remains poorly understood. Here we show that during acute gastrointestinal infection, priming of monocytes for regulatory function preceded systemic inflammation and was initiated prior to bone marrow egress. Notably, natural killer (NK) cell-derived IFN-γ promoted a regulatory program in monocyte progenitors during development. Early bone marrow NK cell activation was controlled by systemic interleukin-12 (IL-12) produced by Batf3-dependent dendritic cells (DCs) in the mucosal-associated lymphoid tissue (MALT). This work challenges the paradigm that monocyte function is dominantly imposed by local signals after tissue recruitment, and instead proposes a sequential model of differentiation in which monocytes are pre-emptively educated during development in the bone marrow to promote their tissue-specific function.

The aged nonhematopoietic environment impairs natural killer cell maturation and function.

Natural killer (NK) cells are critical in eliminating tumors and viral infections, both of which occur at a high incidence in the elderly. Previous studies showed that aged NK cells are less cytotoxic and exhibit impaired maturation compared to young NK cells. We evaluated whether extrinsic or intrinsic factors were responsible for the impaired maturation and function of NK cells in aging and whether impaired maturation correlated with functional hyporesponsiveness. We confirmed that aged mice have a significant decrease in the frequency of mature NK cells in all lymphoid organs. Impaired NK cell maturation in aged mice correlated with a reduced capacity to eliminate allogeneic and B16 tumor targets in vivo. This could be explained by impaired degranulation, particularly by mature NK cells of aged mice. Consistent with impaired aged NK cell maturation, expression of T-bet and Eomes, which regulate NK cell functional maturation, was significantly decreased in aged bone marrow (BM) NK cells. Mixed BM chimeras revealed that the nonhematopoietic environment was a key determinant of NK cell maturation and T-bet and Eomes expression. In mixed BM chimeras, NK cells derived from both young or aged BM cells adopted an ‘aged’ phenotype in an aged host, that is, were hyporesponsive to stimuli in vitro, while adopting a ‘young’ phenotype following transfer in young hosts. Overall, our data suggest that the aged nonhematopoietic environment is responsible for the impaired maturation and function of NK cells. Defining these nonhematopoietic factors could have important implications for improving NK cell function in the elderly.

PTEN Regulates Natural Killer Cell Trafficking in Vivo

IntroductionPhosphatase and tensin homolog (PTEN) is the principal negative regulator of the PI3-kinase pathway, members of which are essential for regulating natural killer (NK) cell activation and effector functions (Tassi et al Immunity 2007, Kim et al Blood 2007). However, the role of PTEN in NK cell biology remains unknown, in part hampered by the embryonic lethality of global PTEN loss. Thus, we hypothesized that disruption of PTEN would uniquely impact NK cell developmental and functional pathways. To evaluate whether PTEN was required for normal NK cell functions, we generated and evaluated a mouse model of NK cell-specific PTEN deficiency (Ncr1iCre knockin x PTENflox; PTENΔ/Δ).ResultsIn contrast to T and B lymphocytes with conditional PTEN loss, we discovered that PTEN primarily acts to regulate NK cell distribution, but not their development. PTEN deletion resulted in a significant loss of NK cells (Fig. 1) in the bone marrow (48% reduction, p<0.001), spleen (56% reduction, p<0.001), and other lymphoid tissues, but markedly increased numbers within the peripheral blood (3.4-fold increase, p<0.001) and lung (3.8-fold increase, p<0.05). Surprisingly, we observed unaltered NK cell maturation (defined by CD27 and CD11b expression) within the peripheral organs of PTENΔ/Δ mice, indicating that PTEN operates to re-distribute NK cells without effects on terminal maturation.To determine whether this aberrant localization could be attributed to dysregulated migration, we examined NK cell trafficking between lymphoid organs. PTEN-deficient NK cells egress more efficiently from the bone marrow and preferentially reside in sinusoidal compartments (mean sinusoidal fraction: 19% [control] vs. 34% [PTENΔ/Δ], p<0.01). Following combined in vivo inhibition of CXCL12 and VLA-4, the peripheral blood of control mice phenocopied the blood NK cell expansion observed in PTENΔ/Δ mice, suggesting that insensitivity to these retention signals is one contributing mechanism. Short-term, i.v. adoptive transfer of control and PTENΔ/Δ NK cells revealed that PTENΔ/Δ NK cells are strongly retained once in the blood (2.8-fold greater retention of blood PTENΔ/Δ vs. control NK cells, p<0.05) (Fig. 2). Furthermore, the loss of PTEN results in increased NK cell homeostatic proliferation in vivo (mean 3-day bone marrow NK cell BrdU incorporation: 20% [control] vs. 52% [PTENΔ/Δ], p=0.0008). Thus, PTEN regulates multiple factors that contribute to the normal steady-state distribution in the naïve mouse.Given the inappropriate localization without PTEN, we further evaluated the NK cell requirement for PTEN during an anti-lymphoma response. PTENΔ/Δ mice challenged i.p. with the NK-sensitive RMA/S lymphoma had defective expansion of the peritoneal compartment (absolute peritoneal NK cells at 48 hours: 1.9x105 vs. 7.2x104, p=0.04). Furthermore, using an adoptive transfer model that requires NK cell trafficking to distal sites of lymphoma challenge, we found that PTEN-deficient NK cells had significant defects in their recruitment to localized tumors (18.4-fold vs 1.5-fold increase in recruited NK cells, p<0.001) (Fig. 3).ConclusionsIn this study, we describe the first report of NK-cell intrinsic PTEN loss in vivo. Collectively, our data suggests that unopposed PI3K signaling in NK cells dominantly affects key events responsible for appropriate cell trafficking and distribution, which is distinct from the role of PTEN in related lymphocyte lineages. These data implicate PTEN as a critical mediator of NK cell recruitment to sites of lymphoma and suggest that PTEN dysregulation, as in the case of PTEN loss-of-function mutations and hamartoma tumor syndromes, may result in defective NK cell-mediated immunity. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Natural killer cell development and maturation in aged mice

The effect of aging on natural killer cell homeostasis is not well studied in humans or in animal models. We compared natural killer (NK) cells from young and aged mice to investigate age-related defects in NK cell distribution and development. Our findings indicate aged mice have reduced NK cells in most peripheral tissues, but not in bone marrow. Reduction of NK cells in periphery was attributed to a reduction of the most mature CD11b+ CD27− NK cells. Apoptosis was not found to explain this specific reduction of mature NK cells. Analysis of NK cell development in bone marrow revealed that aged NK cells progress normally through early stages of development, but a smaller percentage of aged NK cells achieved terminal maturation. Less mature NK cells in aged bone marrow correlated with reduced proliferation of immature NK cells. We propose that advanced age impairs bone marrow maturation of NK cells, possibly affecting homeostasis of NK cells in peripheral tissues. These alterations in NK cell maturational status have critical consequences for NK cell function in advanced age: reduction of the mature circulating NK cells in peripheral tissues of aged mice affects their overall capacity to patrol and eliminate cancerous and viral infected cells.

Nfil3-independent lineage maintenance and antiviral response of natural killer cells

Development of the natural killer (NK) cell lineage is dependent on the transcription factor Nfil3 (or E4BP4), which is thought to act downstream of IL-15 signaling. Nfil3-deficient mice lack NK cells, whereas other lymphocyte lineages (B, T, and NKT cells) remain largely intact. We report the appearance of Ly49H-expressing NK cells in Nfil3(-/-) mice infected with mouse cytomegalovirus (MCMV) or recombinant viruses expressing the viral m157 glycoprotein. Nfil3(-/-) NK cells at the peak of antigen-driven expansion were functionally similar to NK cells from infected wild-type mice with respect to IFN-γ production and cytotoxicity, and could comparably produce long-lived memory NK cells that persisted in lymphoid and nonlymphoid tissues for >60 d. We demonstrate that generation and maintenance of NK cell memory is an Nfil3-independent but IL-15-dependent process. Furthermore, specific ablation of Nfil3 in either immature NK cells in the bone marrow or mature peripheral NK cells had no observable effect on NK cell lineage maintenance or homeostasis. Thus, expression of Nfil3 is crucial only early in the development of NK cells, and signals through activating receptors and proinflammatory cytokines during viral infection can bypass the requirement for Nfil3, promoting the proliferation and long-term survival of virus-specific NK cells.

Differential chemotactic receptor requirements for NK cell subset trafficking into bone marrow

Responsiveness of maturing natural killer (NK) cells to chemotactic molecules directly affect their retention and relocation in selected bone marrow (BM) microenvironment during development, as well as their localization at sites of immune response during inflammatory diseases. BM is the main site of NK cell generation, providing microenvironmental signals required to sustain cell proliferation and differentiation. Drastic changes of expression and function of several chemoattractant receptors can be observed during progression from precursor NK cells to immature and mature NK cells. Indeed, the gradual decrease of CXCR4 expression parallels the increased expression of CXCR3, CCR1, and CX3CR1 and S1P5 (Sphingosine-1-phosphate receptor 5) on mature DX5+ NK cells. The chemokine CXCL12 is produced constitutively in the BM and, acting via CXCR4, is critical for retaining immature and mature NK cell subsets in the BM. During steady-state, the maintenance of NK cells into BM parenchyma depends on the equilibrium of CXCR4 retention and S1P5 mobilizing functions, as the gradient of S1P coming from the sinusoids facilitates mature NK cell egress into circulation via S1P5, when CXCR4/CXCL12-mediated retention decreases. Chemoattractants are also key factors for the response to inflammatory or infection conditions that promote mobilization of effector NK cells from storage compartments (including BM) to sites of disease or for NK cell recruitment/response during pathological conditions that affect BM integrity, including hematopoietic malignancies. In this review, we summarize what is known about the requirement for NK cell localization and exit from BM and how chemokine-mediated functions may affect BM NK cell development and immune responses.

Single line or parallel lines: NK cell differentiation driven by T-bet and Eomes

Single line or parallel lines: NK cell differentiation driven by T-bet and Eomes

Identification of an NK/T cell–restricted progenitor in adult bone marrow contributing to bone marrow– and thymic-dependent NK cells

AbstractAlthough bone marrow (BM) is the main site of natural killer (NK)–cell development in adult mice, recent studies have identified a distinct thymic-dependent NK pathway, implicating a possible close link between NK- and T-cell development in adult hematopoiesis. To investigate whether a potential NK-/T-lineage restriction of multipotent progenitors might take place already in the BM, we tested the full lineage potentials of NK-cell progenitors in adult BM. Notably, although Lin−CD122+NK1.1−DX5− NK-cell progenitors failed to commit to the B and myeloid lineages, they sustained a combined NK- and T-cell potential in vivo and in vitro at the single-cell level. Whereas T-cell development from NK/T progenitors is Notch-dependent, their contribution to thymic and BM NK cells remains Notch-independent. These findings demonstrate the existence of bipotent NK-/T-cell progenitors in adult BM.

Reduced DNAM-1 expression on bone marrow NK cells associated with impaired killing of CD34+ blasts in myelodysplastic syndrome

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal stem-cell disorders characterized by ineffective hematopoiesis and risk of progression to acute myeloid leukemia. Increased apoptosis and suppressed functions of peripheral blood natural killer (NK) cells have been described in MDS patients, but only limited information is available on the phenotypic and functional integrity of NK cells in the bone marrow. In a cohort of 41 patients with distinct clinical subtypes of MDS, we here show that NK cells in the bone marrow show decreased surface expression of the activating receptors DNAM-1 and NKG2D. Notably, decreased receptor expression correlated with elevated bone marrow blast counts and was associated with impaired NK-cell responsiveness to stimulation with the K562 cell line, or co-activation by NKG2D or DNAM-1 in combination with the 2B4 receptor. Furthermore, antibody-masking experiments revealed a central role for DNAM-1 in NK cell-mediated killing of freshly isolated MDS blasts. Thus, given the emerging evidence for NK cell-mediated immune surveillance of neoplastic cells, we speculate that reduced expression of DNAM-1 on bone marrow NK cells may facilitate disease progression in patients with MDS.

Disruption of the meprin α and β genes in mice alters homeostasis of monocytes and natural killer cells

Meprin metalloproteases are implicated in inflammatory bowel disease, which involves dysfunction of immune cells. However, the roles of meprins in the immune and hematological system remain uncharacterized. In this report, we demonstrate that meprins were expressed in the hematological system, and meprin alpha/beta null (alpha(-/-)/beta(-/-)) mice had decreased prevalence of resident monocytes and natural killer (NK) cells in blood, with a concomitant accumulation of inflammatory monocytes and NK cells in bone marrow. In contrast, T and B lymphocytes were not affected by meprin deficiency. In response to acute inflammation induced by intraperitoneal injection of thioglycollate, meprin-deficient mice exhibited higher body temperature than wild-type mice, which was correlated with retention of inflammatory monocytes, but persistent low prevalence of NK cells in blood. These results indicate that meprin metalloproteases play important roles in the homeostasis of monocytes and NK cells, and possibly are involved in egress of these two type cells from bone marrow and homing to the periphery. Our findings are the first report to demonstrate that metalloproteases affect homeostasis of leukocytes, which have important implications for understanding physiology of and pathogenesis in the hematological system.

Systemic Delivery of Nanoliposomal Ceramide Displays Anti-Leukemic Activity in a Rat Model of Large Granular Lymphocytic Leukemia

We have previously demonstrated that enhanced survival rather than increased proliferation accounts for the accumulation of natural killer (NK) cells in large granular lymphocyte (LGL) leukemia patients. To further elucidate the mechanism by which NK survival is enhanced, we analyzed leukemic NK cells isolated either from patient peripheral blood or rat splenic cells for altered expression of members of the inhibitor of apoptosis protein (IAP) family, which act as suppressors of apoptosis in a variety of human solid tumors and hematological malignancies. We now report that the IAP, survivin, was highly expressed in NK cells. In contrast, survivin was barely detectable in NK cells from the blood of normal human donors or the splenic cells from normal rats. We next asked if the lipid-derived second messenger, ceramide, which selectively induces apoptosis in cancer cells would diminish survivin protein expression. Treatment of NKL, a human NK-LGL leukemia cell line, or RNK-16, a rat NK-LGL leukemia cell line, with the cell-permeable, short-chain C6-ceramide (C6) in a pegylated liposomal formulation, led to cell apoptosis and diminished survivin protein expression, in a time and dose dependent manner. We next extended these in vitro studies to an in vivo rat model of NK-LGL leukemia. Systemic i.v. delivery of liposomal ceramide displayed significant anti-leukemia activity in a syngeneic Fischer F344 rat NK-LGL leukemia model that exhibits clonal expansion of CD3-CD8a+ lymphocytes. Over a 6-week treatment period, a well-tolerated dose of 40 mg/kg liposomal-C6, three times a week, elicited a 3 to 10- fold reduction in the weight of various lymphoid and non-lymphoid organs, compared with liposomal formulations that did not contain ceramide (ghost). Untreated or ghost-treated leukemic rats presented with lymphocytosis (LGL counts between 2 × 1011 and 3.5 × 1011/L), anemia and thrombocytopenia. Furthermore, the percentage of NK LGL cells, defined as CD3-CD8a+ by flow cytometry, was drastically elevated in the spleen, lymph node, thymus, bone marrow, blood, liver and lung in these leukemic rats, compared with their normal counterparts. In contrast, leukemic rats treated with liposomal ceramide had undetectable LGL cells in the blood and normal counts of red blood cells and platelets. Additionally, the CD3-CD8a+ NK cells in spleen, thymus and liver were found to be remarkably decreased, whereas the NK cells in bone marrow, blood and lung were within normal range. Collectively, these results indicate that bioactive ceramide analogues can be incorporated into pegylated liposomal vehicles for anti-leukemic efficacy in a rat model of NK LGL leukemia, possibly via decreased survivin expression or signaling.

Bone marrow and peripheral blood natural killer cell activity in lymphomas. Its response to IL-2.

Natural killer (NK) cytotoxic activity was simultaneously investigated in bone marrow mononuclear cells (BMMC) and peripheral blood lymphocytes (PBL) from nine Hodgkin's disease (HD) and 15 non-Hodgkin lymphoma (NHL) untreated patients. Twenty-five PBL samples and seven bone marrow specimens from healthy individuals were also included as control group (C). NK cell activity was evaluated in basal condition and post-stimulation with human recombinant IL-2 (rIL-2). Data were expressed in K values (number of BMMC or PBL needed to lyse 50% of the target cells). In basal condition, both HD and NHL patients showed a NK cell activity comparable to the C group, both in BMMC (HD, K = 2.48 +/- 1.3; NHL, K = 3.8 +/- 2.0; C, K = 3.2 +/- 0.7) and PBL (HD, K = 2.0 +/- 1.0; NHL, K = 2.3 +/- 1.0; C, K = 2.2 +/- 0.2). Stimulation with rIL-2 induced a significant and comparable enhancement of the NK activity in PBL from HD, NHL and C while the response to rIL-2 of the BMMC in most of the HD and NHL patients was significantly greater than the C group. Responder cells were characterized by negative selection with specific MoAb plus complement as a CD3-, CD16+, CD56+ cytotoxic cell and further confirmed by flow cytometry. We postulate that IL-2 activation of bone marrow NK cell precursors, in addition to enhancing the activity of circulating NK, may be of value for the therapeutic rationale of IL-2 in patients with lymphoma.

P110γ and p110δ Phosphoinositide 3-Kinase Signaling Pathways Synergize to Control Development and Functions of Murine NK Cells

Phosphoinositide 3-kinases (PI-3Ks) are key enzymes for cell development, activation, and survival. Here we showed that PI-3K class IB and class IA catalytic subunits, p110gamma and p110delta, played a crucial role in the development and functions of murine NK cells. p110gamma deficiency and impairment of G protein-coupled receptor (GPRC) signaling prevented full NK cell maturation. Concomitant loss of p110gamma and p110delta exacerbated this defect, resulting in a very small population of NK cells with a highly immature phenotype in the bone marrow and periphery. Moreover, combined p110gamma and p110delta signals were required for cytotoxicity and activation of the kinase ERK during NK cell-target cell interaction. p110gamma played a major role in receptor-induced interferon-gamma (IFN-gamma) production through a pathway that involved the kinase ERK and 5-Lipoxigenase, which most likely generates lipid mediators activating GPRCs. Conversely, PI3Ks negatively regulated interleukin-12 (IL-12) and IL-18-induced IFN-gamma by modulating p38 kinase activation. Our data shed light on the multiple intersecting pathways through which PI3Ks control NK cell-mediated innate responses.