In vitro regulation of human hematopoiesis by natural killer cells: analysis at a clonal level

We studied the effects of a series of well-characterized clones of human natural killer (NK) cells on the proliferation of highly purified normal marrow hematopoietic progenitor cells. Individual NK clones suppressed granulocyte, monocyte, erythroid, or mixed colony formation in a heterogeneous but clonally stable manner. Inhibition of colony growth required a period of close cell contact between NK cell and progenitor cell with maximum inhibition occurring after 8 to 18 hours of preincubation time. The mechanism of killing was at least partially humoral, however, as cell-free supernatants generated by NK clones "activated" by contact with a target cell also inhibited progenitor cell growth. One of the possible humoral mediators was identified as gamma-interferon by studies with specific neutralizing monoclonal antibodies. These results show that clonal NK lines can be further activated by coming in contact with hematopoietic progenitor cells, resulting in substantial inhibition of colony formation in vitro.     

Selective blockade of human natural killer cells by a monoclonal antibody.

A murine monoclonal antibody, 13.1, which blocks human natural killer (NK) cell-mediated lysis, has been developed. Hybridoma 13.1 was derived by fusion of NS-1 cells with spleen cells from mice immunized with an enriched population of NK cells. Supernatants of growing hybridomas were screened for their ability to block NK cell-mediated lysis of K562 targets. Antibody 13.1 is an IgG1 with a single light chain type and it does not fix complement. The 13.1 antigen is expressed on all peripheral blood mononuclear cells, with an antigen density approximately 1/30th that of HLA antigen heavy chain. Pretreatment and washing experiments revealed that inhibition of cytotoxicity occurred at the effector cell level only. Significant blocking was achieved with nanogram quantities of antibody and was not due to toxic effects on NK cells. Likewise, controls with other antibodies of the same subclass demonstrated that blocking was not a consequence of mere binding to NK cells. When a panel of 17 NK cell-susceptible targets was tested, the lysis of only 5 of these was blocked, namely K562, HL-60, KG-1, Daudi, and HEL, a human erythroleukemic cell line. The lysis of 12 human B cell and T cell line targets was not inhibited. In addition to the demonstration that the 13.1 antigen is a crucial cell surface structure involved in NK lysis, a heterogeneity of target cell recognition has been revealed that argues for the proposition that individual NK cells have multiple recognitive capabilities.     

Regulation of hematopoiesis by T lymphocytes and natural killer cells

T lymphocytes and natural killer (NK) cells exert both stimulatory and suppressive effects that regulate growth and differentiation of hematopoietic cells. Activated T and NK cells have been demonstrated in different pathological states of bone marrow failure and are proposed to play a role in the pathogenesis of the disease. T and NK cells have also been shown to be responsible for bone marrow graft rejection in both allogeneic and syngeneic donor/recipient combinations. Lymphocytes can regulate hematopoietic cell growth by direct cellular contact or by releasing soluble factors, such as colony-stimulating factors, immune interferon, lymphotoxin, and tumor necrosis factor, active on hematopoietic precursor cells.     

Characterization of a monoclonal antibody against a differentiation antigen of human natural killer cells (NK cells)]

The new murine monoclonal antibody BL-(H5) reacts with a novel surface molecule which is mainly expressed on human NK and B cells. The antigen is not expressed on peripheral T lymphocytes, thymocytes and different human T-cell lines. BL-(H5) does not bind to erythrocytes and platelets. The monoclonal antibody reacts in western blotting experiments with an antigen of 78kDa.     

Mice lacking flt3 ligand have deficient hematopoiesis affecting hematopoietic progenitor cells, dendritic cells, and natural killer cells

The ligand for the receptor tyrosine kinase fms-like tyrosine kinase 3 (flt3), also referred to as fetal liver kinase-2 (flk-2), has an important role in hematopoiesis. The flt3 ligand (flt3L) is a growth factor for hematopoietic progenitors and induces hematopoietic progenitor and stem cell mobilization in vivo. In addition, when mice are treated with flt3L immature B cells, natural killer (NK) cells and dendritic cells (DC) are expanded in vivo. To further elucidate the role of flt3L in hematopoiesis, mice lacking flt3L (flt3L-/-) were generated by targeted gene disruption. Leukocyte cellularity was reduced in the bone marrow, peripheral blood, lymph nodes (LN), and spleen. Thymic cellularity, blood hematocrit, and platelet numbers were not affected. Significantly reduced numbers of myeloid and B-lymphoid progenitors were noted in the BM of flt3L-/- mice. In addition a marked deficiency of NK cells in the spleen was noted. DC numbers were also reduced in the spleen, LN, and thymus. Both myeloid-related (CD11c(++) CD8alpha(-)) and lymphoid-related (CD11c(++) CD8alpha(+)) DC numbers were affected. We conclude that flt3L has an important role in the expansion of early hematopoietic progenitors and in the generation of mature peripheral leukocytes.     

Inhibition of in vitro natural killer activity by the third component of complement: role for the C3a fragment.

Purified human native third component of complement, C3, was found to inhibit in vitro natural killer (NK) cell cytotoxicity in both mouse and human systems. The effect was dose and time dependent, a 50% inhibition being reached with 190 nM C3 (35 micrograms/ml) added during the NK assay or after a 30-min preincubation of the effector cells with this C3 concentration. C3 was shown to act at the effector-cell population level because pretreatment of the target cells did not modify the NK lysis. The inhibition was not due to general cytotoxicity nor to cell agglutination. Moreover, another in vitro cytotoxicity system (represented by alloreactive cytotoxic lymphocytes) was not affected by purified C3. Structural analysis of the active part of the C3 molecule shows that the C3-induced inhibition is supported by the C3a fragment. Release of carboxyl-terminal arginine residue by carboxypeptidase B, converting C3a into des-Arg77-C3a, did not alter the inhibitory effect displayed by this fragment. These results suggest that C3a may play an important role in the regulation of NK activity.     

Agranulocytosis induced by propylthiouracil: evidence of a drug dependent antibody reacting with granulocytes, monocytes and haematopoietic progenitor cells

A patient with agranulocytosis and myeloid marrow hypoplasia following a second exposure to propylthiouracil (PTU) was studied for antibodies against mature blood cells and bone marrow precursor cells. During the acute phase of the agranulocytosis, significant growth inhibition of the myeloid committed progenitor cells (CFU-GM) was found following incubation with complement, indicating the presence of in-vivo cell bound cytotoxic antibodies. Using immunofluorescence and complement dependent cytotoxicity techniques it was demonstrated that acute phase and recovery phase sera contained circulating antibodies, reactive not only with differentiated granulocytes and monocytes but also with myeloid and erythroid (BFU-E/CFU-E) progenitor cells. Complement dependent lysis of the progenitor cells was facilitated by preincubation with PTU. These results indicate that the agranulocytosis was mediated by a PTU dependent antibody that affected both mature blood cells and bone precursor cells.     

Enhancement of ligand-dependent activation of human natural killer T cells by lenalidomide: therapeutic implications

Natural killer T (NKT) cells are CD1d-restricted glycolipid reactive innate lymphocytes that play an important role in protection from pathogens and tumors. Pharmacologic approaches to enhance NKT cell function will facilitate specific NKT targeting in the clinic. Here we show that lenalidomide (LEN), a novel thalidomide (Thal) analog, enhances antigen-specific expansion of NKT cells in response to the NKT ligand alpha-galactosylceramide (alpha-GalCer) in both healthy donors and patients with myeloma. NKT cells activated in the presence of LEN have greater ability to secrete interferon-gamma. Antigen-dependent activation of NKT cells was greater in the presence of dexamethasone (DEX) plus LEN than with DEX alone. Therapy with LEN/Thal also led to an increase in NKT cells in vivo in patients with myeloma and del5q myelodysplastic syndrome. Together these data demonstrate that LEN and its analogues enhance CD1d-mediated presentation of glycolipid antigens and support combining these agents with NKT targeted approaches for protection against tumors.     

In vitro inhibition of hematopoiesis by HNK1, DR-positive T cells and monocytes after allogeneic bone marrow transplantation

Blood CFU-GM and BFU-E, grown from 17 patients who had undergone allogeneic bone marrow transplantation (BMT), were studied by the plasma-clot technique before day 45, and at a time when their blood count was approximately normal. The number of colonies varied from one patient to another, but was always lower than in normal subjects. Removal of cells forming rosettes with sheep erythrocytes (E+C) increased colony growth in four out of eight cases, whereas removal of adherent cells (AC) had the same effect in five out of six cases. Addition of E+C or AC after their initial removal restored the inhibition of colony growth. This suppression was noted at a 1:1 to 8:1 cellular ratio and ranged from 25% to 75%. The phenotype of the suppressive cells was further characterized by complement-mediated lysis with monoclonal antibodies (MoAbs) and fluorescent labeling. Two types of cells associated with inhibition of colony growth were identified: the first were E+C positive, characterized by the T3, HNK1, DR, and T8 determinants; the second were identified by the MO2 MoAb, indicating their monocytic origin, together with their properties of adherence. Similar suppressor cells of CFU-GM were found in the marrow of two other allogeneic BMT patients. A direct suppressive effect of the two types of cells was demonstrated in one experiment when MO2+ and/or HNK1+ cells collected by cell sorting were added back to cultures depleted in MO2+ and HNK1+ cells by complement-mediated lysis and were both found to decrease colony growth. Purified HNK1+ cells led to moderate inhibition of colony growth, which was not enhanced by increasing their concentration. This suppressive effect of hematopoiesis could be the consequence of an allogeneic reaction, since no inhibition was affected by T cells or monocytes in seven autologous BMTs and one syngeneic BMT.     

In vivo evidence for a dependence on interleukin 15 for survival of natural killer cells

Cellular homeostasis requires a balance between cell production, cell survival, and cell death. Production of natural killer (NK) cells from bone marrow precursor cells requires interleukin 15 (IL-15); however, very little is known about the factors controlling survival of mature NK cells in vivo. Because mice deficient in IL-15 (IL-15(-/-) mice) fail to develop NK cells, it is not known whether mature NK cells can survive in an environment lacking IL-15. We hypothesized that IL-15 might indeed be required for survival of mature NK cells in vivo. Freshly isolated NK cells labeled with 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE) were adoptively transferred into IL-15(-/-) mice and littermate control (IL-15(+/-)) mice. Within 36 hours after transfer, NK cells were detected in both IL-15(-/-) and IL-15(+/-) mice; however, significantly more (P <.003) CFSE-positive (CFSE(+)) NK cells were found in control mice than in IL-15(-/-) mice. By 5 days, similar numbers of CFSE(+) NK cells were still easily detected in IL-15(+/-) mice, whereas no CFSE(+) NK cells survived in IL-15(-/-) mice. Furthermore, mice with severe combined immunodeficiency treated with the Fab fragment of a blocking antibody recognizing a signaling subunit of the IL-15 receptor, IL-2/15Rbeta, had a significant ( approximately 90%) loss of NK cells compared with control mice. Finally, NK cells from Bcl-2 transgenic mice that were adoptively transferred into IL-15(-/-) mice did survive. These results show conclusively that IL-15 is required for mature NK cell survival in vivo and suggest that IL-15 mediates its effect on NK cell survival by means of Bcl-2.     

Production of interferons by dendritic cells, plasmacytoid cells, natural killer cells, and interferon-producing killer dendritic cells.

The capacity of mouse spleen conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) to produce interferon-gamma (IFN-gamma) or IFN-alpha was assessed, and compared with that of natural killer (NK) cells and the recently identified interferon-producing killer dendritic cells (IKDCs), both of which are frequent contaminants in DC preparations. Fully developed cDCs or pDCs, if free of NK cells or IKDCs, showed little capacity for IFN-gamma production. However, an early developmental form of the CD4-8+ cDC subtype, and the Ly6C- Ly49Q- pDC subtype, both were able to produce moderate amounts of IFN-gamma, although less than IKDCs. In response to toll-like receptor 9 stimuli, both the Ly6C+ Ly49Q+ and the Ly6C- Ly49Q- pDC subtypes were effective producers of IFN-alpha. However, IKDCs, which efficiently produced IFN-gamma and showed immediate cytotoxicity on NK target cells, did not produce IFN-alpha under these conditions.     

Neutrophil-endothelial cell interaction: evidence in vitro for a regulation by endothelial cells of neutrophil functions.

The purpose of this study was to investigate a possible relationship between human umbilical vein endothelial cells (EC) triggered by ionophore A23187 at different doses (0.5-2.5 microM) and polymorphonuclear neutrophils (PMN). EC supernatants were shown to contain neutrophil chemoattractant activity (NCA) and in parallel a factor inducing an inhibition of PMN chemiluminescence (PMN CL). Supernatants obtained from EC triggered by A23187 exhibited a high level of NCA (73 +/- 5 PMN.hpf-1 compared to 21 +/- 4 PMN.hpf-1 in untreated EC supernatants, p less than 0.01). This NCA was independent from arachidonic acid metabolites, since indomethacin and nordi-hydroguaiaretic acid failed to suppress the chemotactic activity. Using gel filtration chromatography (AcA 54) the NCA was recovered in a single peak of apparent molecular weight of 37,000 +/- 4,000 daltons. Checkerboard analysis indicated that NCA exhibited both chemotactic and chemokinetic activities. In addition, supernatants of A23187-stimulated EC, and at a lesser degree, supernatants of unstimulated EC, inhibited PMN CL induced by N-formyl-Methionyl-Leucyl-Phenylalanine (61% inhibition, p less than 0.05), and by A23187 itself (80% inhibition, p less than 0.01), but not that induced by phorbol-myristate-acetate. Indomethacin and protamine sulphate did not modulate this inhibitory activity. By contrast, EC-derived inhibitory activity was inhibited (50%) by an adenosine antagonist (8-phenyltheophylline), indicating a participation of adenosine in this inhibitory activity of PMN CL. These data suggest the possibility that activated endothelial cells could both enhance PMN migration and protect themselves against potential damaging effects of oxygen metabolites produced by PMN, particularly during transvascular migration.     

Interferon gamma encapsulated into liposomes enhances the activity of monocytes and natural killer cells and has antiproliferative effects on tumor cells in vitro

Summary The effects of human interferon gamma (IFN gamma) encapsulated into liposomes were investigated in vitro. Monocytes were induced to release a cytotoxic factor with either IFN gamma encapsulated into liposomes, free IFN gamma or lipopolysaccharide (LPS). If IFN gamma was applied in the liposomal form, less IFN activity was required to stimulate monocytes. Most of the cytotoxic factor was secreted during the first 4 h of stimulation. The cytotoxic factor in supernatants from PMNLs was completely neutralized by a monospecific polyclonal antiserum to tumor necrosis factor (TNF). Combining subthreshold doses of IFN gamma liposomes or IFN gamma with lipopolysaccharide synergistically enhanced the release of TNF. In fluorescence analysis, altered expression of the class II HLA-DR antigen on LeuM3 positive monocytes was induced with IFN gamma liposomes as well as with IFN gamma. Not only monocytes but also natural killer (NK) cells were stimulated to higher cytotoxicity by IFN gamma liposomes in a dose-dependent manner. In comparison with IFN gamma, the same amount of activity was necessary for adequate stimulation of NK-cells against the K562 target cells. Furthermore, the antiproliferative effects of IFN gamma liposomes and free IFN gamma on several human tumor cell lines was compared. Among several cell lines tested, U937 and A549 turned out to be sensitive to IFN gamma, and both cell lines reacted with 50% growth inhibition at a lower amount of gamma presented by liposomes than in the free form. These data show production of IFN gamma liposomes which possess immunmodulatory and antiproliferative activity in vitro. In several of the test systems studied, liposome-encapsulated IFN gamma was more effective than free IFN gamma.     

Platelet activation by immobilized monoclonal antibody: evidence for a CD9 proximal signal.

Anti-CD9 monoclonal antibodies (MoAbs) are reported to activate human platelets through stimulation of the Fc gamma II receptor. We show here that nonstimulatory F(ab')2 fragments of the anti-CD9 MoAb 50H.19 induce dense-granule release and dose-dependent platelet aggregation when attached to polystyrene latex beads. Cross-linking F(ab')2 fragments of MoAb 50H.19 by F(ab')2 fragments of goat anti-mouse IgG does not result in platelet aggregation unless the second antibody is bound to latex beads, indicating that immobilization, and not cross-linking of the stimulus, is critical to the initiation of the CD9 signal. In contrast, F(ab')2 fragments of the second antibody readily induce the aggregation of platelets treated with the anti-Fc gamma II receptor MoAb IV.3. Immobilization of MoAb per se is insufficient to induce an activation signal because intact and F(ab')2 fragments of nonstimulatory MoAb directed to glycoprotein Ib and HLA class I do not become stimulatory when attached to beads. CD9-induced activation requires cytoskeletal rearrangement because it is inhibited by cytochalasin B. Aggregation is blocked by inhibitors of the thromboxane pathway, indicating that CD9 activates phospholipase C indirectly through prior activation of phospholipase A2.