Induce Natural Killer Cell Research Articles

KIR-LIGAND Mismatched Natural Killer Cells Effectively Kill Primary Myeloma and Myeloma Cell Lines.

Recent observations in haplo-identical, T-cell-depleted allogeneic transplantation have focused attention on the rapid and remarkable anti-tumor effect mediated by Killer Inhibitory Receptor (KIR) ligand mismatched natural killer (NK) cells. Most data are confined to the role of KIR-ligand mismatched NK cells for acute leukemia after allogeneic transplantation. In this study, we report both the ability of KIR mismatched NK cells to kill primary myeloma cells, and the effects of interleukin (IL)2 and IL15 on NK cells in vitro. NK cells were prepared from healthy donor PBMCs by depletion of T-lymphocytes using immunomagnetic beads conjugated to anti-CD3 followed by depletion of monocytes using a simple adherence step. After overnight incubation in cytokines (IL2/IL15 as indicated) standard chromium release assays determined the cytotoxicity of NK cells against target cells and 3H-thymidine incorporation assessed NK cell proliferation. KIR-ligand mismatched NK cells incubated overnight in 300 IU/ml IL2 killed primary MM cells from four individuals (76, 64,60,48% lysis) as well as MM lines U266, NCI H929, and the NK sensitive line K562 (95, 86, 76% lysis, respectively) at an E:T ratio of 20:1. NK cells were not cytotoxic towards autologous phytohemagglutinin (PHA) or allogeneic PHA-induced T-cell blasts. Increasing the IL2 concentration during overnight NK cell incubation from 300 to 1000 IU/ml did not significantly enhance the cytotoxicity against U266 myeloma cells or control K562 cells. IL15 was more potent than IL2 in inducing NK cell proliferation. The optimal IL15 concentration was 300 IU/ml (range tested: 0–3000 IU/ml). The use of both cytokines in concert was less effective than the use of IL15 alone, probably due to homology of the IL2 and IL15 b and g receptors. We conclude that T cell and monocyte depletion of PBMC results in a preparation significantly enriched for NK cells (±50%) that effectively kills KIR-ligand mismatched primary myeloma cells and myeloma cell lines. IL15 is the superior cytokine for enhancing NK cell proliferation. The exciting anti-leukemic effects mediated by KIR-ligand mismatched NK cells have thus far only been reported in the haplo-identical transplant setting. In view of our positive in vitro data in MM, we will evaluate in a phase II trial wheter the repeated transfusion of KIR-ligand-mismatched, T-cell depleted NK cells from haplo-identical donors after immunosuppressive treatment with fludarabine and dexamethasone (to avoid rejection of donor NK cells) and tumor reduction with melphalan followed by a delayed auto-transplant can improve outcome in patients who have high risk MM or who have relapsed after a previous auto-transplant. This will be the first clinical application of KIR-ligand-mismatched NK cells in autologous transplantation and the first such trial in myeloma. [Display omitted]

MECHANISMS OF BACILLUS CALMETTE-GUERIN MEDIATED NATURAL KILLER CELL ACTIVATION

Natural killer (NK) cells are of crucial importance for bacillus Calmette-Guerin (BCG) mediated antitumor effects. We defined the mechanisms of BCG mediated NK cell activation in vitro. A standard Cr release assay was used to measure the cytotoxicity of BCG activated NK cells. Using the MACS system (Miltenyi Biotec, Bergisch-Gladbach, Germany) we depleted various immune cell subpopulations from BCG stimulated peripheral blood mononuclear cells to phenotype activated NK cells. During the stimulation process anticytokine antibodies and recombinant cytokines were added to define their role in NK cell activation. For costimulation studies peripheral blood mononuclear cells were separated into lymphocytes and monocytes by counterflow-centrifugation (elutriation). Inhibitory NK cell receptor expression on activated NK cells was measured by flow cytometry by antiCD3, antiCD56 and anti-inhibitory NK cell receptor triple staining. The accessory function of monocytes was indispensable for BCG mediated NK cell activation. However, the stimulatory potential of monocytes did not require direct cell-cell contact to NK cells or major histocompatibility complex dependent antigen presentation to T cells. Monocyte derived interleukin (IL)-12 and to a lesser extent interferon (IFN)-alpha were key mediators for stimulating BCG induced NK cell cytotoxicity and IFN-gamma production. In contrast, IL-10 inhibited NK cell cytotoxicity and IL-18 did not show any effect. Exogenous recombinant IFN-alpha and IL-12 enhanced BCG mediated secretion of IFN-gamma and yet BCG induced NK cell cytotoxicity remained unchanged. While the CD158a and CD158b subsets did not have a significant role, NKG2A cells represented the predominant cytolytic subset in BCG activated NK cells. Following BCG stimulation the monocyte derived TH1 cytokines IL-12 and IFN-alpha activate tumor cytotoxic CD3/CD56/NKG2A NK cells. Our results elucidate NK activating mechanisms that are operative during BCG immunotherapy for bladder cancer and are relevant for an early, innate antimycobacterial immune response.

Stimulation of natural killer (NK) cell and macrophage infiltration in pancreatic cancer with virulizin (V), an immunotherapeutic agent

4257 Purpose: V is stimulates macrophages and monocytes in vitro and in vivo. Pancreatic cancer patients treated with V in phase I/II studies showed increased 6 and 12 months survival vs historical controls. V administration was associated with increased NK cell activity in patients showing the best response to V. Hence, NK cell function may be a mediator of V anti-tumor activity. Methods: To determine if V induces NK cell infiltration into tumors, CD-1 nude mice with Capan-1 pancreatic tumor xenografts were treated with V; tumor samples were analyzed by flow cytometry using NK cell-specific antibodies. Anti-tumor activity and NK cell infiltration were also evaluated in human tumor xenografts in CD-1 nude mice depleted of macrophages. Immunohistochemical staining of tumor xenografts was performed to further assess NK cell and macrophage activity. Results: V increased NK cell infiltration of tumors (59.4 – 116.0%). Macrophage depletion reduced both NK cell infiltration of tumors, and anti-tumor activity of V (P=0.1632). V-treated mice showed a significant increase in infiltration of F4/80+ (macrophages) and NK1.1+ (NK) cells, vs saline treated controls. Increased NK1.1+ cell infiltration occurred early in V treatment, and correlated with an early marker for tumor apoptosis. V treatment also resulted in a significant increase in the NK cell number in the spleen. Conclusion: Macrophage-mediated NK cell activation/recruitment is implicated in the mechanism of action of V.. These results suggest that V mediates a sustained infiltration of NK cells and macrophages into tumors. Low NK cell activity may be a risk factor for malignancy and metastases, and a negative prognostic indicator in pancreatic cancer. NK cell function and other immune activities are being assessed as biologic markers of clinical response in a Phase III trial in pancreatic cancer as a first line combination therapy with gemcitabine. NK cell function will be correlated with clinical response parameters. No significant financial relationships to disclose.

Stimulation of natural killer (NK) cell and macrophage infiltration in pancreatic cancer with virulizin (V), an immunotherapeutic agent

4257 Purpose: V is stimulates macrophages and monocytes in vitro and in vivo. Pancreatic cancer patients treated with V in phase I/II studies showed increased 6 and 12 months survival vs historical controls. V administration was associated with increased NK cell activity in patients showing the best response to V. Hence, NK cell function may be a mediator of V anti-tumor activity. Methods: To determine if V induces NK cell infiltration into tumors, CD-1 nude mice with Capan-1 pancreatic tumor xenografts were treated with V; tumor samples were analyzed by flow cytometry using NK cell-specific antibodies. Anti-tumor activity and NK cell infiltration were also evaluated in human tumor xenografts in CD-1 nude mice depleted of macrophages. Immunohistochemical staining of tumor xenografts was performed to further assess NK cell and macrophage activity. Results: V increased NK cell infiltration of tumors (59.4 – 116.0%). Macrophage depletion reduced both NK cell infiltration of tumors, and anti-tumor activity of...

Role of natural killer cells in innate protection against lethal ebola virus infection.

Ebola virus is a highly lethal human pathogen and is rapidly driving many wild primate populations toward extinction. Several lines of evidence suggest that innate, nonspecific host factors are potentially critical for survival after Ebola virus infection. Here, we show that nonreplicating Ebola virus-like particles (VLPs), containing the glycoprotein (GP) and matrix protein virus protein (VP)40, administered 1–3 d before Ebola virus infection rapidly induced protective immunity. VLP injection enhanced the numbers of natural killer (NK) cells in lymphoid tissues. In contrast to live Ebola virus, VLP treatment of NK cells enhanced cytokine secretion and cytolytic activity against NK-sensitive targets. Unlike wild-type mice, treatment of NK-deficient or -depleted mice with VLPs had no protective effect against Ebola virus infection and NK cells treated with VLPs protected against Ebola virus infection when adoptively transferred to naive mice. The mechanism of NK cell–mediated protection clearly depended on perforin, but not interferon-γ secretion. Particles containing only VP40 were sufficient to induce NK cell responses and provide protection from infection in the absence of the viral GP. These findings revealed a decisive role for NK cells during lethal Ebola virus infection. This work should open new doors for better understanding of Ebola virus pathogenesis and direct the development of immunotherapeutics, which target the innate immune system, for treatment of Ebola virus infection.

Cytolytic capabilities of lamina propria and intraepithelial lymphocytes in normal and chronically inflamed human intestine.

Cell-mediated lymphocyte cytotoxicity in ileum and colon of patients with ulcerative colitis (UC), Crohn's disease (CD) and controls was investigated. Frequencies of cells expressing perforin and Fas-ligand (FasL) were determined by immunomorphometry. mRNA expression of perforin, granzyme B and FasL in T cells and subsets was assayed by reverse transcriptase-polymerase chain reaction. Cytotoxicity of intraepithelial and lamina propria lymphocytes was analysed without ex vivo activation in three functional assays: (1) anti-CD3-dependent T-cell receptor (TCR)-/CD3-mediated redirected cytotoxicity, (2) Fas-/FasL-mediated TCR-/CD3-independent cytotoxicity and (3) natural killer (NK) cell cytotoxicity. Inflammation in ileum of CD patients caused increased frequency of perforin-expressing cells and enhanced perforin-dependent TCR-/CD3-mediated cytotoxicity. In contrast, lymphocytes in the inflamed colon of UC or Crohn's colitis patients did not display this cytotoxicity nor did lymphocytes of normal colon. Normal colon lymphocytes showed spontaneous Fas-/FasL-mediated cytotoxicity. This activity was retained but not enhanced in inflamed UC colon. In contrast, a significant increase of FasL-expressing cells was seen in situ. Inflammation did not induce NK cell activity in colonic lymphocytes. Intestinal lymphocytes comprise effectors active in two different cytolytic processes. 'Classical' cytotoxic T lymphocytes in small intestine and lymphocytes executing TCR-/CD3-independent FasL-/Fas-mediated killing of unknown biological role present throughout the intestinal mucosa. Ongoing normal cytolytic processes seem to be enhanced by chronic inflammation.

Induction of potent antitumour natural-killer cells from peripheral blood of patients with advanced prostate cancer.

To examine whether antitumour natural-killer (NK) cells can be induced from peripheral blood mononuclear cells (PBMCs) of patients with advanced prostate cancer, as cell therapy using antitumour immune cells is a promising candidate treatment but such patients generally have a suppressed immune response against cancer cells. PBMCs were obtained from 10 patients (four with stage D2 and six with stage B or C disease). For the NK cell expansion, PBMCs were co-cultured with irradiated HFWT cells, a cell line originating from Wilms' tumour, in RHAM alpha culture medium supplemented with 5% autologous plasma and interleukin-2 (200 U/mL) for 2 weeks. When PBMCs were co-cultured with HFWT cells, lymphocytes from all patients had a 20- to 130-fold expansion after 2 weeks of culture. The CD16+ CD56+ cells constituted >70% of the proliferated lymphocyte population. The induced NK cells had significantly greater cytotoxicity against a prostate cancer cell line (PC-3) than lymphocytes cultured with no HFWT cells. There was no significant difference in growth and phenotypes of lymphocytes and the induced NK cell activity between patients with stage D2, B or C. NK cells with potent cytotoxic activity against prostate cancer cell lines from patients with advanced prostate cancer were selectively expanded. Further investigation is needed to determine whether this approach could be a candidate for cell therapy for advanced prostate cancer.

Soluble HLA class I induces NK cell apoptosis upon the engagement of killer-activating HLA class I receptors through FasL-Fas interaction

The engagement of the activating isoforms of C-type lectin inhibitory receptor (CLIR) or killer Ig-like receptor (KIR) by their natural ligands, represented by soluble HLA-I (sHLA-I) molecules, induced programmed cell death of natural killer (NK) cells. Indeed, NK cell apoptosis elicited by either putative HLA-E and HLA-F (sHLA-I non-A, -B, -C, and -G) or sHLA-I-Cw4 or -Cw3 from untransfected or -Cw4 or -Cw3 alleles transfected HLA-A(-), B(-), C(-), G(-), E(+), F(+) 721.221 lymphoblastoid cell line, respectively, was blocked by covering the corresponding activating receptor with either anti-CLIR- or anti-KIR-specific monoclonal antibodies (mAbs). After sHLA-I-activating receptor interaction, NK cells produced and released Fas ligand (FasL), which in turn led to NK cell apoptosis by interacting with Fas at the NK cell surface. Blocking anti-Fas mAb, or anti-FasL mAb, inhibited sHLA-I-mediated apoptosis via activating receptor in NK cell clones. This apoptosis was inhibited by NK cell treatment with cyclosporin A, whereas this drug had no effect on activating receptor-mediated activation of cytolysis. Conversely, concanamycin A, an inhibitor of vacuolar type H(+)-adenosine triphosphatase (H(+)-ATPase) of granules, inhibited activating receptor-induced NK cell cytolysis, suggesting that activating receptor-mediated apoptosis and cytolysis can use different intracellular pathways. Furthermore, a large amount of interferon-gamma (IFN-gamma) was detectable in culture supernatant of activating receptor(+) NK cells incubated with the appropriate sHLA-I ligand. Again, cyclosporin A, but not concanamycin A, strongly reduced activating receptor-mediated IFN-gamma production. This suggests that activating receptor-induced apoptosis of NK cells could play a role in eliminating potentially harmful NK cell clones and, at the same time, it leads to production of IFN-gamma, an antiviral cytokine able to amplify immune responses.

Partial impairment of interleukin-12 (IL-12) and IL-18 signaling in Tyk2-deficient mice

AbstractTyk2 is activated in response to interleukin-12 (IL-12) and is essential for IL-12–induced T-cell function, including interferon-γ (IFN-γ) production and Th1 cell differentiation. Because IL-12 is a stimulatory factor for natural killer (NK) cell–mediated cytotoxicity, we examined whether tyk2 is required for IL-12–induced NK cell activity. IL-12–induced NK cell activity in cells from tyk2-deficient mice was drastically reduced compared to that in cells from wild-type mice. IL-18 shares its biologic functions with IL-12. However, the molecular mechanism of IL-18 signaling, which activates an IL-1 receptor–associated kinase and nuclear translocation of nuclear factor-κB, is different from that of IL-12. We next examined whether biologic functions induced by IL-18 are affected by the absence of tyk2. NK cell activity and IFN-γ production induced by IL-18 were reduced by the absence of tyk2. Moreover, the synergistic effect of IL-12 and IL-18 for the production of IFN-γ was also abrogated by the absence of tyk2. This was partially due to the absence of any up-regulation of the IL-18 receptor treated with IL-12, and it might suggest the presence of the cross-talk between Jak-Stat and mitogen-activated protein kinase pathways in cytokine signaling.

Systemic administration of liposome-encapsulated OK-432 prolongs the survival of rats with hepatocellular carcinoma through the induction of IFN-gamma-producing hepatic lymphocytes.

OK-432 is known to increase the host antitumor response. We previously reported that systemic administration of OK-432 (OK-Lipo) specifically induced hepatic lymphocytes in mice. Here we aimed to investigate the antitumor effect of OK-Lipo on hepatocellular carcinomas (HCC) in experimental rats. Diethylnitrosamine was administered for 12 weeks to all rats (n = 36). Rats were divided into three groups of 12 rats each. One group was injected with OK-Lipo from week 5 (OK-5w group) and another from week 9 (OK-9w group). A control group was injected with saline from week 5 (Non-OK group). At week 13, five rats from each group were used for histological analysis and immunofluorescence assays (surface phenotypic and intracellular cytokine analysis of the mononuclear cells in the liver, spleen and peripheral blood). The remaining rats were observed for the remainder of their survival period. The mean survival times of Non-OK, OK-5w, and OK-9w groups differed significantly (98.0 +/- 5.3 days, 116.0 +/- 5.8 days, and 106.0 +/- 5.4 days, respectively, P < 0.01). Histological examination revealed many apoptotic tumor cells, infiltration of lymphocytes and macrophages in the OK-5w group. The two-color immunofluorescence assay showed that the proportion of natural killer (NK) cells and IFN-gamma-producing cells in the liver were significantly higher in the OK-5w group. These findings showed that systemic administration of OK-Lipo contributed to prolonging the survival of rats with HCC. OK-Lipo induced NK cells and IFN-gamma-producing cells specifically in the liver and these cells seemed to reduce hepatocarcinogenesis and tumor growth.

A novel role for interleukin-18 in human natural killer cell death: high serum levels and low natural killer cell numbers in patients with systemic autoimmune diseases.

Patients with systemic autoimmune diseases have been reported to have reduced numbers of peripheral blood natural killer (NK) cells compared with healthy subjects. The ability of selected cytokines to trigger NK cell death prompted us to compare the levels of peripheral blood cytokines with the numbers of NK cells in patients with various systemic autoimmune diseases. We used enzyme-linked immunosorbent assays to measure the concentration of selected cytokines (interleukin-18 [IL-18], IL-15, IL-12, IL-2, interferon-gamma [IFNgamma], and tumor necrosis factor alpha [TNFalpha]) in sera from 58 patients with systemic autoimmune diseases and 33 healthy controls. The absolute number of T cells and NK cells in the peripheral blood was measured in parallel using flow cytometry. The ability of selected cytokines to induce NK cell death was then measured using 3,3'-dihexyloxacarbocyanine iodide dye, propidium iodide staining, and caspase 3 activity. Levels of IL-18, IL-15, IFNgamma, and TNFalpha were elevated in sera from patients with systemic autoimmune diseases compared with normal controls. The percentage of NK cells and natural killer T cells were significantly decreased in the peripheral blood of patients with systemic autoimmune diseases compared with normal controls. Serum concentrations of IL-18, IL-15, and TNFalpha were inversely related to the number of NK cells in both patients and healthy controls. The combination of IL-18 and IL-15 or IL-18 and IL-12 induced NK cell death in vitro. The combination of IL-18 and IL-15 or IL-18 and IL-12 enhanced IFNgamma and TNFalpha production by NK cells in vitro. Cytokine-induced NK cell death is caspase-dependent and is partially blocked by neutralizing antibodies against TNFalpha. High levels of IL-18 and IL-15 are associated with the decreased number of NK cells that is observed in patients with systemic autoimmune diseases.

Natural killer cell dysfunction and apoptosis induced by chronic myelogenous leukemia cells: role of reactive oxygen species and regulation by histamine

Natural killer (NK) cells are deficient in patients with chronic myelogenous leukemia (CML), but the mechanisms responsible for the dysfunction are not completely understood. This study reports that CML cells effectively inhibit the baseline and interleukin-2 (IL-2)-induced NK cell cytotoxicity against a CML cell-derived line (K562). A sizable fraction of NK cells subsequently acquired features characteristic of programmed cell death/apoptosis. The CML cell-mediated inhibition of NK cells required triggering of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated formation of reactive oxygen species (ROS) and was prevented by catalase, a scavenger of ROS, and by histamine, acting via H2-receptor–mediated inhibition of ROS production in CML cells. In contrast, nonmalignant neutrophilic granulocytes inhibited NK cells via ROS production without the requirement of exogenous NADPH oxidase-triggering stimuli. We propose that paracrine production of ROS may contribute to the dysfunction of NK cells in CML and that histamine may serve as an autocrine inhibitor of ROS formation in leukemic granulocytes.

Deconstructing the KIR–HLA complex

1 Boyington, J.C. et al. (2000) Crystal Structure of an NK cell immunoglobulin-like receptor in complex with its class I MHC ligand. Nature 405, 537–5432 Tormo, J. et al. (2000) Crystal structure of a lectin-like natural killer receptor bound to its MHC class I ligand. Nature 402, 623–6313 Natarajan, K. et al. (1999) Interaction of the NK cell inhibitory receptor Ly49A with H-2Dd: identification of a site distinct from the TCR site. Immunity 11, 591–601When major histocompatibility complex (MHC) class I molecules present foreign peptides to cytotoxic CD8+ T cells, the T cells are activated to kill the target cells. Natural killer (NK) cells also have receptors that interact with MHC class I molecules, but interaction with class I/self-peptides generates an inhibitory signal that prevents NK cell lysis of the target cell. Thus, NK cells do not kill normal, healthy class I-expressing cells, but do kill cells that express inadequate levels of class I, such as cancer cells. Inhibitory receptors on NK cells belong to two families – the immunoglobulin (Ig) superfamily (called killer inhibitory receptors, KIR), and the C-type lectin superfamily (called Ly49 receptors). Human inhibitory NK receptors can be of either type, whereas mice only have Ly49 receptors.Now, Boyington et al. report the first crystal structure of a KIR (KIR2DL2) in complex with its class I ligand (HLA-Cw3)1. The structure reveals that dimeric KIR binds across the top of the α1 and α2 helices of HLA-Cw3, and interacts with amino acids at positions 7 and 8 of the peptide. This interaction is similar to that between the T-cell receptor (TCR) and MHC class I. However, the TCR binds centrally across the groove whereas the KIR binds at one end of the groove, with an area of overlap in-between. For NK T cells, this overlap suggests that it is unlikely that a TCR and a KIR on the same NK cell can simultaneously interact with a single MHC class I molecule.The KIR2DL2/HLA-Cw3 interaction is very different to the interaction between the murine dimeric receptor Ly49A and its class I ligand H-2Dd, which was described recently2,3. In this case, a single Ly49 molecule binds at one side of the peptide-binding groove. Although Ly49 does not interact directly with the peptide, the peptide must fill the groove for the Ly49A/H-2Dd interaction to occur.These studies provide the first structural information on the interaction between NK inhibitory receptors and MHC class I ligands. Further studies are required to address the role of zinc ions in KIR/HLA interactions, and to determine how KIR variants that induce NK cell lysis interact with class I molecules.

Recruitment and activation of natural killer (NK) cells in vivo determined by the target cell phenotype. An adaptive component of NK cell-mediated responses.

Natural killer (NK) cells can spontaneously lyse certain virally infected and transformed cells. However, early in immune responses NK cells are further activated and recruited to tissue sites where they perform effector functions. This process is dependent on cytokines, but it is unclear if it is regulated by NK cell recognition of susceptible target cells. We show here that infiltration of activated NK cells into the peritoneal cavity in response to tumor cells is controlled by the tumor major histocompatibility complex (MHC) class I phenotype. Tumor cells lacking appropriate MHC class I expression induced NK cell infiltration, cytotoxic activation, and induction of transcription of interferon gamma in NK cells. The induction of these responses was inhibited by restoration of tumor cell MHC class I expression. The NK cells responding to MHC class I-deficient tumor cells were approximately 10 times as active as endogenous NK cells on a per cell basis. Although these effector cells showed a typical NK specificity in that they preferentially killed MHC class I-deficient cells, this specificity was even more distinct during induction of the intraperitoneal response. Observations are discussed in relation to a possible adaptive component of the NK response, i.e., recruitment/activation in response to challenges that only NK cells are able to neutralize.

Regulation of Human Natural Killer Cell Migration and Proliferation by the Exodus Subfamily of CC Chemokines

Natural killer (NK) cells play an important role in innate and adaptive immune responses to obligate intracellular pathogens. Nevertheless, the regulation of NK cell trafficking and migration to inflammatory sites is poorly understood. Exodus-1/MIP-3α/LARC, Exodus-2/6Ckine/SLC, and Exodus-3/MIP-3β/ELC/CKβ-11 are CC chemokines that share a unique aspartate-cysteine-cysteine-leucine motif near their amino terminus and preferentially stimulate the migration of T lymphocytes. The effects of Exodus chemokines on human NK cells were examined. Exodus-1, -2, and -3 did not induce detectable chemotaxis of resting peripheral blood NK cells. In contrast, Exodus-2 and -3 stimulated migration of polyclonal activated peripheral blood NK cells in a dose-dependent fashion. Exodus-2 and -3 also induced dose-dependent chemotaxis of NKL, an IL-2-dependent human NK cell line. Results of modified checkerboard assays indicate that migration of NKL cells in response to Exodus-2 and -3 represents true chemotaxis and not simply chemokinesis. Exodus-1, -2, and -3 did not induce NK cell proliferation in the absence of other stimuli. Nevertheless, Exodus-2 and -3 significantly augmented IL-2-induced proliferation of normal human CD56dim NK cells. In contrast, Exodus-1, -2, and -3 did not affect the cytolytic activity of resting or activated peripheral blood NK cells. Expression of message for CCR7, a shared receptor for Exodus-2 and -3, was detected in activated polyclonal NK cells and NKL cells but not resting NK cells. Taken together, these results indicate that Exodus-2 and -3 can participate in the recruitment and proliferation of activated NK cells. Exodus-2 and -3 may regulate interactions between T cells and NK cells that are crucial for the generation of optimal immune responses.

Indications of the protective role of natural killer cells in human cutaneous leishmaniasis in an area of endemicity.

The role of natural versus acquired immunity to Leishmania aethiopica infection in humans is the focus of our studies. We found in previous studies that mononuclear cells from nonexposed healthy Swedish donors responded to Leishmania antigen stimulation by proliferation and gamma interferon production. The main cell type responding was CD3- CD16/56+ natural killer (NK) cells. These findings led us to suggest that the potential to produce a rapid, nonacquired NK cell response may be a protective phenotype. In order to test this hypothesis, an area in Ethiopia where Leishmania is endemic was selected, and peripheral blood mononuclear cells were obtained from individuals who had lived in the area most of their lives but had no evidence of past or present leishmaniasis. Their responses were compared with those of confirmed leishmaniasis patients from the same region with active lesions or cured leishmaniasis lesions. Cells from these donors were stimulated in vitro with L. aethiopica antigen. Responses were measured by proliferation, cytokine production, and phenotype analysis by fluorescence-activated cell sorting. The association of NRAMP1 alleles with the studied phenotype and susceptibility to L. aethiopica-induced leishmaniasis was also evaluated. The results show that Leishmania antigens can induce NK cell and CD8+-T-cell responses in vitro. This is clearly seen in proliferating cells from the cured (immune) individuals and the apparently protected controls from the area of endemicity. It contrasted with the reactivity of the patients, where some NK proliferation was coupled with enhanced CD4+-T-cell proliferation. We conclude from these observations that NK cells and CD8(+) cells proliferating in response to Leishmania stimulation are involved in protection from and healing of (Ethiopian) cutaneous leishmaniasis; however, such mechanisms appear to be unrelated to the NRAMP1 host resistance gene.

Stem Cell Factor Enhances Interleukin-2–Mediated Expansion of Murine Natural Killer Cells In Vivo

AbstractThe administration of low dose interleukin-2 (IL-2) results in a selective expansion of natural killer (NK) cells in vivo, and promotes the differentiation of NK cells from hematopoietic precursor cells in vitro. We have previously shown that stem cell factor (SCF ), the ligand to the c-kit tyrosine kinase receptor, enhances IL-2–induced NK cell proliferation and differentiation in vitro. Here, we investigated the effects of SCF plus IL-2 delivered to mice in vivo. Eight-week-old C57BL/6 mice were treated with a continuous subcutaneous infusion of IL-2 (1 × 104 IU/d) plus a daily intraperitoneal dose of SCF (100 μg/kg/d), IL-2 alone, SCF alone, or vehicle alone for 8 weeks. The in vivo serum concentration of IL-2 ranged between 352 ± 12.0 pg/mL and 606 ± 9.0 pg/mL, achieving selective saturation of the high affinity IL-2 receptor, while the peak SCF serum concentration was 296 ± 13.09 ng/mL. Alone, the daily administration of SCF had no effect on the expansion of NK cells. The continuous infusion of IL-2 alone did result in a significant expansion of NK1.1+CD3− cells compared to mice treated with placebo or SCF. However, mice treated with both SCF and IL-2 showed an increase in the absolute number of NK cells that was more than twofold that seen with IL-2 alone, in the spleen (P ≤ .005), bone marrow (P ≤ .025), and blood (P < .05). NK cytotoxic activity against YAC-1 target cells was significantly higher for mice treated with SCF plus IL-2, compared to mice treated with IL-2 alone (P ≤ .0005). Interferon-γ (IFN-γ) production in cytokine-activated splenocytes was also greater for the SCF plus IL-2 group, over IL-2 treatment alone (P ≤ .01). The effect of SCF plus IL-2 on NK cell expansion was likely mediated via NK cell precursors, rather than mature NK cells. In summary, we provide the first evidence that SCF can significantly enhance expansion of functional NK cells induced by the prolonged administration of low dose IL-2 in vivo. Since the NK cell is a cytotoxic innate immune effector and a potent source of IFN-γ, this therapeutic strategy for NK cell expansion may serve to further enhance innate immune surveillance against malignant transformation and infection in the setting of cancer and/or immunodeficiency.

Virus-induced transient bone marrow aplasia: major role of interferon-alpha/beta during acute infection with the noncytopathic lymphocytic choriomeningitis virus.

The hematologic consequences of infection with the noncytopathic lymphocytic choriomeningitis virus (LCMV) were studied in wild-type mice with inherent variations in their interferon (IFN)-alpha/beta responder ability and in mutant mice lacking alpha/beta (IFN-alpha/beta R0/0) or gamma IFN (IFN-gamma R0/0) receptors. During the first week of infection, wild type mice demonstrated a transient pancytopenia. Within a given genetic background, the extent of the blood cell abnormalities did not correlate with the virulence of the LCMV isolate but variations were detected between different mouse strains: they were found to depend on their IFN-alpha/beta responder phenotype. Whereas IFN-gamma R0/0 mice were comparable to wild-type mice, IFN-alpha/beta R0/0 mice exhibited unchanged peripheral blood values during acute LCMV infection. In parallel, the bone marrow (BM) cellularity, the pluripotential and committed progenitor compartments were up to 30-fold reduced in wild type and IFN-gamma R0/0, but remained unchanged in IFN-alpha/beta R0/0 mice. Viral titers in BM 3 d after LCMV infection were similar in these mice, but antigen localization was different. Viral antigen was predominantly confined to stromal BM in normal mice and IFN-gamma R0/0 knockouts, whereas, in IFN-alpha/beta R0/0 mice, LCMV was detected in > 90% of megakaryocytes and 10-15% of myeloid precursors, but not in erythroblasts Although IFN-alpha/beta efficiently prevented viral replication in potentially susceptible hematopoietic cells, even in overwhelming LCMV infection, unlimited virus multiplication in platelet and myeloid precursors in IFN-alpha/beta R0/0 mice did not interfere with the number of circulating blood cells. Natural killer (NK) cell expansion and activity in the BM was comparable on day 3 after infection in mutant and control mice. Adaptive immune responses did not play a major role because comparable kinetics of LCMV-induced pancytopenia and transient depletion of the pluripotential and committed progenitor compartments were observed in CD8(0/0) and CD4(0/0) mice, in mice depleted of NK cells, in lpr mice, and in perforin-deficient (P0/0) mice lacking lytic NK cells. Thus, the reversible depression of hematopoiesis during early LCMV infection was not mediated by LCMV-WE-specific cytotoxic T lymphocyte, cytolysis, or secreted IFN-gamma from virally induced NK cells but was a direct effect of IFN-alpha/beta.

Role of Interleukin-2 (IL-2), IL-7, and IL-15 in Natural Killer Cell Differentiation From Cord Blood Hematopoietic Progenitor Cells and From γc Transduced Severe Combined Immunodeficiency X1 Bone Marrow Cells

Natural killer (NK) cells are characterized by their ability to mediate spontaneous cytotoxicity against susceptible tumor cells and infected cells. They differentiate from hematopoietic progenitor cells. Patients with X-linked severe combined immunodeficiency (SCID X1) carry mutations in the gamma c cytokine receptor gene that result in lack of both T and NK cells. To assess the role of interleukin-2 (IL-2), IL-7, and IL-15 cytokines, which share gamma c receptor subunit, in NK cell differentiation, we have studied NK cell differentiation from cord blood CD34 (+) cells in the presence of either stem cell factor (SCF), IL-2, and IL-7 or SCF and IL-15. The former cytokine combination efficiently induced CD34 (+) CD7 (+) cord blood cells to proliferate and mature into NK cells, while the latter was also able to induce NK cell differentiation from more immature CD34 (+) CD7 (-) cord blood cells. NK cells expressed CD56 and efficiently killed K562 target cells. These results show that IL-15 could play an important role in the maturation of NK cell from cord blood progenitors. Following retroviral-mediated gene transfer of gamma c into SCID X1 bone marrow progenitors, it was possible to reproduce a similar pattern of NK cell differentiation in two SCID-X1 patients with SCF + IL-2 + IL-7 and more efficiently in one of them with SCF + IL-15. These results strongly suggest that the gamma c chain transduces major signal(s) involved in NK cell differentiation from hematopoietic progenitor cells and that IL-15 interaction with gamma c is involved in this process at an earlier step than IL-2/IL-7 interactions of gamma c are. It also shows that gene transfer into hematopoietic progenitor cells could potentially restore NK cell differentiation in SCID X1 patients.

Involvement of Cytokine Gene Expression in the Age-Dependent Decline of NK Cell Response

The molecular mechanism responsible for the age-dependent decline of natural killer (NK) cell response was examined. The responses of NK cells to poly(I:C), Con A, and LPS were compared between the young and the aged animals. We observed that both basal and induced NK cell activity declined with age. To further determine the basis for this change, an effort was made to identify the cytokine genes that might be involved. In addition, the capacity of acetyl-L-carnitine (ALC) to restore NK cell activity was evaluated. In this study,51Cr release assay and reverse transcriptase–polymerase chain reaction assay were used to measure the NK cell activity and the changes in cytokine gene expression, respectively. Our results show that the decline in NK cell response to poly(I:C) and LPS in aged animals was correlated with decreased expression of the IFN-γ gene. The decline in the NK cell response to Con A in aged animals was, however, independent of the expression of IL-2 or IFN-γ genes. Although ALC was reported to restore functions of T cells and macrophages in aged animals, we found that long-term treatment (4 months) with ALC had no effect on either the basal or the induced age-dependent loss of NK cell activity. Our observations suggest that cytokine gene therapy could be a potential approach to improving or even preventing the decline of certain immune functions in the elderly.