Natural Killer Cells In Bone Marrow Research Articles

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.

Association of bone marrow natural killer cell dose with neutrophil recovery and chronic graft‐versus‐host disease after HLA identical sibling bone marrow transplants

Allogeneic bone marrow (BM) transplant (BMT) outcomes have been correlated with the infused nucleated, CD34(+), and T- cell dose. The potential impact of natural killer (NK) BM infused cell dose has however not been established. We analysed the outcomes of 78 patients receiving an HLA identical BMT. A higher NK cell dose was associated with the speed of neutrophil (P = 0.05) and platelet recovery (P = 0.04). Higher nucleated cells, CD34(+), CD3(+), CD3(+)/4(+), CD3(+)/8(+) and NK cell dose were associated with a lower incidence of chronic GvHD (cGvHD) in univariate analysis. In multivariate analysis, the risk of cGvHD was increased by a lower NK cell dose [hazard ratio (HR) = 2.3 (1.2-4.4) for cell dose <0.9 x 10(6)/kg; P = 0.01] and an older age [HR = 1.4 /10 years (1.1-1.8); P = 0.002]. In addition, a higher CD3(+)/4(+) and NK cell dose were associated with a decreased incidence of viral infections (P = 0.03 and P = 0.06 respectively). No specific cell subpopulation infused dose was associated with survival. In conclusion, a higher BM NK cell dose is associated with an increased speed of neutrophil recovery and a decreased incidence of cGvHD.

A thymic pathway of mouse natural killer cell development characterized by expression of GATA-3 and CD127

Natural killer (NK) cell development is thought to occur in the bone marrow. Here we identify the transcription factor GATA-3 and CD127 (IL-7R alpha) as molecular markers of a pathway of mouse NK cell development that originates in the thymus. Thymus-derived CD127+ NK cells repopulated peripheral lymphoid organs, and their homeostasis was strictly dependent on GATA-3 and interleukin 7. The CD127+ NK cells had a distinct phenotype (CD11b(lo) CD16- CD69(hi) Ly49(lo)) and unusual functional attributes, including reduced cytotoxicity but considerable cytokine production. Those characteristics are reminiscent of human CD56(hi) CD16- NK cells, which we found expressed CD127 and had more GATA-3 expression than human CD56+ CD16+ NK cells. We propose that bone marrow and thymic NK cell pathways generate distinct mouse NK cells with properties similar to those of the two human CD56 NK cell subsets.

Bcl-x Is Indispensable for the Development of Natural Killer Cells.

The size of the Natural Killer (NK) cell pool is maintained through production and subsequently export from the bone marrow, peripheral survival and proliferation, and ultimately cell death. While there exists considerable knowledge about developmental stages of lymphoid, myeloid and erythroid cells, comparably little is known about NK cell intermediates and the genes required for their development. Most of the models of NK cell differentiation have been based on in vitro culture systems where NK cells could be generated from multipotent HSC precursors. However, this approach suffers from the problems inherent to in vitro cell manipulations. We have utilized conditional gene targeting in adult mice to examine the role of the bcl-x gene in the development of NK cells in bone marrow and after their export to the spleen. Bcl-x is an important member of the anti-apoptotic member of the Bcl-2 gene family, and a critical role for this gene in the survival of hematopoietic cells was demonstrated in bcl-x-deficient mice causing embryonic death due to massive apoptosis of immature hematopoietic cells and of neurons. Conditional deletion in erythroid cells lead to hemolytic anemia and extensive splenomegaly. Furthermore, bcl-x is critical for the maturation of pre-B cells to the pro B cell stage, while it is not essential for the development of effector and memory T cells. We have conditionally deleted the gene in the stem cells of adult mice by cross breeding them with the Mxi-cre deleter strain, which allows for induced expression of cre recombinase by injection with pIpC.As early as 9 days after the first injection of pIpC, the number of NK cells in the bone marrow of mice started to decline as demonstrated by multi-color FACS analysis staining for IL2 Receptor beta (CD122) and NKG2D, among other markers. Cultures of bone marrow and spleen cells in the presence of cytokines to generate lymphokine activated killer cells failed, while no such effect was observed in cultures from Mxi-cre mice that were subjected to pIpC injections and carried along as controls. Analysis of animals after 3 weeks of pIpC administration revealed absence of NK cell precursors in the bone marrow as demonstrated by the lack of CD122+/Lin- negative cells. This phenomenon was accompanied by a reduction in the number of mature NK cells in the spleen.To date, six stages of NK cell maturation are described with the acquisition of IL2 receptor beta expression marking commitment to the NK-cell lineage. IL2 Receptor beta as well as NKG2D are expressed throughout NK cell development and at all stages. In order to characterize the specific stage at which expression of bcl-x is essential for NK cell maturation, we employed multi-color FACS analysis staining for CD122, NKG2D, CD49b, and the integrins CD43 and Mac-1 after depletion of lineage positive cells, followed by sorting for defined populations. Real Time PCR on sorted cells demonstrated that Bcl-x mRNA is highly expressed throughout all stages of NK cell development. Taken together, the gradual reduction in the number of NK cell precursors eventually leading to complete loss of this lineage in the bone marrow and peripheral sites suggests that bcl-x is indispensable for the development of NK cells presumably from the earliest time point of commitment to this lineage.

Recombinant Human Prolactin Improves Antitumor Effects of Murine Natural Killer Cells in vitro and in vivo

Objective: To verify the effect of prolactin on natural killer (NK) cells in vivo and its implications for NK cell immunotherapy. Methods: Recombinant human prolactin (rhPRL; 30 µg, i.p.) was administered to BALB/c mice and severe combined immunodeficiency (SCID) mice 1 day before harvesting splenocytes for ⁵¹Cr release assay to examine the effects of rhPRL on NK cells of normal mice. rhPRL (10 µg, i.p., every other day for a total of 5 injections) was administered to BALB/c mice after syngeneic bone marrow transplantation (SBMT) to determine its effects on NK cell reconstitution. CT26 tumor cells were injected into BALB/c mice intravenously on day 0, and interleukin (IL)-15-cocultured activated syngeneic NK cells (IL-15-NK) from SCID mice were intravenously injected into tumor-bearing BALB/c mice on day 1. The improvement of antimetastasis effects and survival of tumor-bearing mice by rhPRL were checked. Results: BALB/c and SCID mice receiving one rhPRL injection exhibited a significant increase in cellular cytotoxicity against YAC-1 target tumor cells; the specific lysis was enhanced from 12.5 to 17.3% in BALB/c mice and from 27.8 to 51.2% in SCID mice. BALB/c mice continuously receiving rhPRL injections exhibited significant increases in NK cell (DX5-positive) contents and cellularity in both the bone marrow and spleen in the SBMT model. The bone marrow NK cell contents were improved from 1.53 to 3.13% after rhPRL injection. NK cells from SCID mice were then cultured with recombinant human IL-15 (rhIL-15; 6,000 IU/ml), rhPRL (10 ng/ml) or rhIL-15 plus rhPRL for 25 days. The cytotoxicity and cellularity were enhanced by rhPRL when tested on day 10, when comparing the rhIL-15 plus rhPRL group with the rhIL-15 group or rhPRL group, respectively. In the adoptive cellular immunotherapy study, the IL-15-NK plus IL-15 plus rhPRL group showed significantly lower numbers of lung metastases and longer survival than the IL-15-NK plus IL-15 group; the mean survival interval was prolonged from 31.5 to 53.7 days. Conclusion: These results indicate that rhPRL is possibly an important regulator of NK cells and a potential biologic product for immunotherapy.

Differential requirement for the transcription factor PU.1 in the generation of natural killer cells versus B and T cells

AbstractPU.1 is a member of the Ets family of transcription factors required for the development of various lymphoid and myeloid cell lineages, but its role in natural killer (NK) cell development is not known. The study shows that PU.1 is expressed in NK cells and that, on cell transfer into alymphoid Rag2/γc−/−mice, hematopoietic progenitors of PU.1−/−fetal liver cells could generate functional NK cells but not B or T cells. Nevertheless, the numbers of bone marrow NK cell precursors and splenic mature NK cells were reduced compared to controls. Moreover,PU.1−/− NK cells displayed reduced expression of the receptors for stem cell factor and interleukin (IL)-7, suggesting a nonredundant role for PU.1 in regulating the expression of these cytokine receptor genes during NK cell development.PU.1−/− NK cells also showed defective expression of inhibitory and activating members of the Ly49 family and failed to proliferate in response to IL-2 and IL-12. Thus, despite the less stringent requirement for PU.1 in NK cell development compared to B and T cells, PU.1 regulates NK cell differentiation and homeostasis.

Mechanisms accounting for the impaired natural-killer cell activity in refractory anaemia with excess of blasts.

Natural killer (NK) cells were analyzed in 38 untreated patients with refractory anaemia with excess of blasts (RAEB), using cytotoxicity assays and immunofluorescence with monoclonal antibodies. We found that patients with RAEB have normal numbers of peripheral blood and bone marrow NK cells. NK cells from RAEB patients express very low natural-killer cell activity (NKa) which may be increased significantly with recombinant alpha-interferon and recombinant interleukin-2, although it remains below the lower limit of the control range. The cells exhibit normal tumour cell binding capacity, but fail to release sufficient amounts of natural-killer cytotoxic factors (NKCFs) upon their interaction with NK-sensitive K562 cell targets or their stimulation with phytohaemagglutinin. Our results suggest that defective NKa in RAEB patients may be due, at least in part, to impaired release of functionally active NKCFs. This disturbance is probably the result of some intrinsic defect of RAEB NK cells in NKCF production, storage, and/or release. The possibility of an impairment in the activation signal provided by the stimulatory K562 cells cannot be excluded, although it seems unlikely. We postulate that this abnormality might represent a manifestation of dysplastic haemopoiesis. Further studies are certainly needed to investigate whether other defective mechanisms are also implicated in the determination of the low NKa in patients with RAEB.

Effect of interleukin-4 on interleukin-2-dependent generation of natural killer cells

We have previously shown that interleukin-2 (IL-2) is able to induce the generation of natural killer (NK) activity in bone marrow (BM) cells from mice pretreated with 5-fluorouracil. IL-2 alone could dose-dependently induce NK activity in marrow cells and interleukin-4 (IL-4) has dual effect on the NK activity in that, depending on the concentration of IL-2, IL-4 inhibits or stimulates development of NK cells. The inhibitory effect was in part antagonized by interleukin-1α. These effects were not obtained when NK-reactive spleen cells were cultured with the same concentrations of IL-2 or IL-2 plus IL-4 with or without irradiated BM cells as feeders. The effects of IL-4 were also obtained by preincubation for 6–24 hr before culturing with IL-2 alone and correlated with the expression of interleukin-2 receptor (IL-2/r), suggesting that IL-4 might play a regulatory role in the IL-2-dependent generation of NK cells in BM.

The primary role of murine bone marrow in the production of natural killer cells. A cytokinetic study.

The normal steady state production of natural killer (NK) cells in the bone marrow and spleen was characterized with cytokinetic technics. We developed a protocol to enrich for NK cells in bone marrow and demonstrate that target binding can be used as a criterion for marrow NK cells if nonspecifically "sticky" cells are eliminated. The selected population of B cell-depleted bone marrow lymphoid cells was comprised mainly of lymphocytes, of which 80% were NK-1.1+. B cell-depleted bone marrow lymphocytes that bound to YAC-1 could be characterized as two populations on the basis of morphology and proliferative status: large, proliferating target-binding cells (TBC), of which 25% were in S phase of the mitotic cycle, and small postmitotic TBC. Pulse and chase studies indicated that the small TBC in bone marrow were derived from an immediate proliferating precursor, presumably the large TBC, which were, in turn, derived from a precursor population that was more rapidly proliferating. In contrast, few if any splenic TBC were labeled after a 30-min pulse with [3H]TdR and significant numbers of labeled TBC did not appear in the spleen until 2 or more days after the pulse label. Surprisingly, some of the splenic TBC were relatively long lived and survived 2 mo or longer. These studies are the first to directly characterize the production of NK cells in situ in normal marrow. We demonstrate that the marrow is the primary site of production of NK cells and that little, if any, proliferation of NK cells occurs in the periphery of unstimulated mice. The data suggest the existence in the bone marrow of at least three compartments in the NK lineage: a rapidly proliferating NK precursor population, a less rapidly proliferating population of large TBC, and a population of small postmitotic TBC.

Strong natural killer (NK) cell activity in bone marrow of myeloma patients: accelerated maturation of bone marrow NK cells and their interaction with other bone marrow cells.

Bone marrow natural killer (NK) cell activity was studied in patients with multiple myeloma. Bone marrow mononuclear cells from myeloma patients expressed considerable levels of cytotoxicity against K562 in a 4 h 51Cr-release assay, which was comparable to that of blood lymphocytes. In contrast, NK-cell activity was markedly low or absent in bone marrow of normal donors and control patients. Fractionation of bone marrow cells from myeloma patients on linear bovine serum albumin gradients enriched NK effector cells in the upper, lymphocyte-enriched fraction, with no reactivity in the middle fraction containing mainly myeloma cells. Treatment with either Leu-7 or OKMI monoclonal antibody plus complement reduced or abrogated bone marrow NK-cell activity of myeloma patients as well as blood NK-cell activity. However, OKMI plus complement failed to reduce control bone marrow NK-cell activity, although the activity was reduced by Leu-7 plus complement. Overnight exposure to interferon (IFN) of bone marrow cells resulted in an augmentation of NK-cell activity in myeloma patients, but not in controls. Furthermore, adherent bone marrow cells from controls suppressed IFN-induced enhancement of NK-cell activity of blood lymphocytes, whereas bone marrow of myeloma patients did not contain such suppressor cells. No cytotoxicity was induced in control bone marrow cells by cocultivation with bone marrow myeloma cells. Bone marrow NK cells from myeloma patients were able to lyse control bone marrow cells in a 18 h assay and their lytic activity was augmented by IFN treatment. However, neither bone marrow cells nor blood lymphocytes were cytotoxic to autologous and allogeneic fresh myeloma cells even after activation with IFN. These results suggest that NK precursor cells differentiate into HNK-I- and OKMI-positive mature NK cells in bone marrow of myeloma patients, but not in control bone marrow, and that these functional bone marrow NK cells may interact with other bone marrow elements.

Suppression of natural killer activity in human blood and bone marrow cultures by bone marrow-adherent OKM1-positive cells

Maintenance and regulation of natural killer (NK) cell activity in human bone marrow cultures were studied using K562 leukemia cells as targets. Culture of bone marrow cells in medium supporting long-term generation of myeloid cells resulted in a rapid loss of NK activity in 1–3 days. In contrast, antibody-dependent cytotoxicity to an NK-resistant tumor was maintained for more than 7 weeks. Horse serum, a component of the myelopoietic culture medium, was found to diminish NK cytotoxicity of blood and bone marrow cultures whereas hydrocortisone supplement did not. In addition, an adherent cell is present in bone marrow which greatly inhibits NK activity. Nonadherent bone marrow cells exhibited higher cytotoxicity than unfractionated cells at all days of culture; adherent cells were not cytotoxic to K562. Purified adherent marrow cells inhibited the cytotoxic capacity of nonadherent blood or marrow mononuclear cells during coculture. Indomethacin, an inhibitor of protaglandin synthesis, augmented levels of NK activity in cultures of bone marrow cells, indicating that macrophages may be suppressing this effector function via prostaglandins. Further identification of the adherent suppressor cells came from experiments in which suppression was prevented by treatment of the adherent cells with monoclonal OKM1 antibody plus complement. This study shows that bone marrow-adherent OKM1-positive cells, presumably macrophages, negatively regulate NK activity, and it defines conditions for analysis of the generation and/or positive regulation of NK cells in human bone marrow.