Enriched NK Cells Research Articles

Tumoricidal Potential of Native Human Blood Dendritic Cells: Direct Tumor Cell Killing and Activation of NK Cell-Mediated Cytotoxicity.

Dendritic cells (DCs) are characterized by their unique capacity for primary T cell activation, providing the opportunity of DC-based cancer vaccination protocols. Recently, we defined a novel major subset of human blood DCs by using the monoclonal antibody M-DC8 which recognizes a carbohydrate modification of P selectin glycoprotein ligand-1 (PSGL-1) selectively expressed on these cells (Immunity 2002; 17:289-301). In addition to a marked capacity to activate tumor-reactive cytotoxic T cells M-DC8+ DCs efficiently mediate antibody-dependent cytotoxicity (Blood ; 2002; 100:1502-1504). In the present study, we analyzed the capacity of M-DC8+ DCs to kill tumor cells in the absence of antibodies and to enhance the tumor-directed cytotoxicity of NK cells. To determine whether M-DC8+ DCs exhibit tumoricidal activity, DCs were isolated at high purity (>93%) from the blood of healthy donors by immunomagnetic separation. These cells were cultured for 6 h in the presence or absence of 200 U/ml interferon (IFN)-gamma. Subsequently, DCs were coincubated with four chromium-labeled tumor cell lines and two normal cell lines for 18 h. Whereas unstimulated DCs demonstrated only moderate tumor-directed cytotoxicity (specific lysis: 7–13%), IFN-gamma-stimulated M-DC8+ DCs displayed potent killing of each of these tumor cell lines (specific lysis: 27–35%). Only a marginal cytotoxic effect was seen when normal human cells such as lung fibroblasts or endothelial cells were used as targets. When evaluating the cytotoxic effector mechanisms FACS analysis and ELISA assays revealed that IFN-gamma-stimulated M-DC8+ DCs secreted a high amount of tumor necrosis factor (TNF)-alpha induced by direct cell-to-cell contact with the different tumor cell lines. This effect was already observed after 3 h of cocultivation. Interestingly, no significant induction of TNF-alpha was detected during contact of M-DC8+ DCs with the normal human cell lines. These results suggest that tumor-associated surface molecules are important for the observed increase of TNF-alpha production in M-DC8+ DCs. Inhibition experiments with neutralizing antibodies clearly demonstrated that tumor cell-induced TNF-alpha play an important role in tumor-directed cytotoxicity mediated by M-DC8+ DCs. To investigate whether M-DC8+ DCs enhance the tumoricidal activity of NK cells freshly isolated DCs were cultured for 6 h in the presence or absence of IFN-gamma. Thereafter, DCs were coincubated with highly enriched (>90%) NK cells. The cytotoxic potential of the stimulated NK cells was tested towards various tumor cell lines in a 4 h chromium release assay. We observed a two- to threefold increase of NK cell-mediated cytotoxicity towards all analyzed tumor cell lines by IFN-gamma-stimulated M-DC8+ DCs. In addition, transwell experiments demonstrated that this triggering effect was mainly dependent on cell-to-cell contact. In conclusion, our data provide evidence that a major subpopulation of circulating human blood DCs exhibits efficient tumoricidal activity and clearly enhances NK cell-mediated tumor-directed cytotoxicity. The capacity of DCs to induce tumor-specific T cell responses and to kill tumor cells either directly or by activating NK cells points to the pivotal role of DCs in triggering the innate and adaptive immune response against tumors.

Clinical scale isolation of T cell-depleted CD56+ donor lymphocytes in children.

We present a clinical scale method for immunomagnetic separation of CD56+ donor natural killer cells for adoptive immunotherapy of pediatric leukemias after allogeneic transplantation. This time-saving and partially automated procedure employed CD56+ selection followed by CD3+ depletion, resulting in a median purity of 98.6% NK cells and a four-log depletion of T cells. The enriched NK cells demonstrated high cytotoxic activity against K562 target cells and fresh leukemic blasts with low HLA class I expression, which could be further enhanced by IL-2 stimulation. Lysis of NK-insensitive leukemic cells with high HLA class I expression could also be demonstrated via ADCC. Due to the high degree of T cell depletion, alloreactive proliferation in mixed lymphocyte cultures and response to T cell-specific mitogen stimulation was profoundly decreased. Our results suggest that, even in the case of mismatched donors, infusions of donor NK cells with extremely low T cell content may be a promising treatment option for leukemic minimal residual disease after allogeneic transplantation without risk of inducing severe GVHD.

Natural killer cell cytokine production, not cytotoxicity, contributes to resistance against blood-stage Plasmodium chabaudi AS infection.

Our recent study showed that IL-12 treatment of susceptible A/J mice induces Th1-mediated, protective immunity against lethal blood-stage Plasmodium chabaudi AS infection. To further understand the mechanism of this protection, we examined NK cell cytotoxic (NKCC) and cytokine secretory functions in untreated and IL-12-treated A/J mice, along with resistant C57BL/6 (B6) mice. Normal A/J mice receiving six daily doses of 0.1 microg IL-12 exhibited significant increases in NKCC in total spleen cell populations. Defective NKCC evident in vitro in enriched NK cells from infected A/J mice was corrected by addition of 10 ng/ml IL-12 and was comparable with that seen in B6 mice. In vivo and in vitro analyses revealed that enriched NK cells from day 6 infected A/J mice were defective not only in NKCC, but also in IFN-gamma, and to a certain extent, TNF-alpha secretion, which could also be corrected by IL-12 treatment. Depletion of NK cells from resistant B6 mice resulted in a more severe course of infection, while NK cell-depleted, IL-12-treated A/J mice had significantly higher parasitemia, as well as 100% mortality, suggesting the importance of NK cells in IL-12-mediated protection. NKCC-defective bg/bg mice produced optimum IFN-gamma and TNF-alpha and recovered from infection similar to bg/+ controls; in vivo depletion of these cytokines resulted in significantly higher parasitemia early in infection. Based on these results, we conclude that IFN-gamma, and possibly TNF-alpha, secretion by NK cells during early infection plays a major role in protective immunity to blood-stage malaria.

The NK2.1 receptor is encoded by Ly-49C and its expression is regulated by MHC class I alleles.

A dual receptor system composed of activation and inhibitory receptors apparently controls NK cell-mediated lysis. In the C57BL/6 mouse, the NK1.1 molecule acts as an activation receptor whereas Ly-49A, C and G2 can inhibit NK cell lysis of target cells expressing specific MHC class I molecules. We previously reported that NK2.1 is an activation receptor sharing structural properties with members of the NKR-P1 and Ly-49 receptor families. In this study, we have shown that NK2.1 is encoded by the previously described Ly-49C gene. We also found that the expression level of NK2.1/Ly-49C is modulated by H-2-dependent factors and that this regulation differs from that previously described for Ly-49A. Flow cytometry analyses of NK-enriched spleen cells from MHC congenic strains on C57BL/10 and BALB/c backgrounds indeed revealed that the level of NK2.1/Ly-49C expression, but not the number of positive cells, is low in strains expressing H-2b and H-2k haplotypes as compared to H-2d mice. Similar analyses of splenic NK cells from two series of congenic and congenic recombinant strains on the C57BL/10 background indicate that the main regulatory element(s) are most likely the H-2Kb and H-2Dk alleles. Together with our and others previous observations, these results identify the NK2.1/Ly-49C antigen as a receptor for MHC class I molecules whose expression is regulated by host MHC genes.

Allogeneic MHC-mismatched activated natural killer cells administered after bone marrow transplantation provide a strong graft-versus-leukaemia effect in mice.

Allogeneic lymphocytes administered with an unmanipulated bone marrow transplant provide a strong antileukaemic effect, the so-called graft-versus-leukaemia (GVL) effect. On the other hand, T-cell-mediated graft-versus-host-disease (GVHD) observed after transplantation of unmanipulated BM graft causes substantial morbidity and mortality. The aim of the present study was to determine the antileukaemic potential of enriched IL-2 activated NK cells administered 2 h after BMT. Balb/c (H-2d) mice were given a dose of A20 (H-2d, B-cell leukaemia) cells 2 d prior to lethal total body irradiation (TBI) and transplantation of either syngeneic or allogeneic anti-Thy1.2 (CD90) depleted bone marrow cells. Either syngeneic (Balb/c, H-2d) or allogeneic (C57BL/6, H-2b) enriched and IL-2 (200 U/ml for 24 h) activated NK cells were given 2 h after BMT. Injection of A20 leukaemia into normal Balb/c recipients led to death after a median of 14 d. A lethal dose of TBI followed by either syngeneic or allogeneic Thy1.2-depleted BMT resulted in a modest antileukaemic effect. The adoptive transfer of syngeneic enriched and IL-2 preincubated NK cells given at time of BMT exerted a significantly better GVL effect. However, the infusion of allogeneic enriched NK cells resulted in a stronger GVL effect. These results clearly demonstrate that allogeneic NK cells are superior to syngeneic NK cells in their potential to eradicate residual leukaemia cells after BMT without mediating clinical overt GVHD. This experimental setting may offer a strategy for treatment of haematological malignancies in a phase of minimal residual disease.

IFN-gamma-inducing factor up-regulates Fas ligand-mediated cytotoxic activity of murine natural killer cell clones.

Abstract NK cells, non-T non-B immune effector lymphocytes, are localized in many organs, including liver, as well as in the circulation. To investigate the regulatory mechanism of killing apparatus in hepatic NK cells, we established IL-2-dependent NK cell clones from liver lymphocytes of BALB/c nude mice. To generate the NK cell clones, we incubated liver lymphocytes with a high dose of IL-2 in the presence of irradiated Kupffer cells, as feeder cells and as the source of IL-12, originally identified as NK cell stimulatory factor. Unless liver lymphocytes were incubated with both IL-2 and Kupffer cells, no cell growth was observed. Hepatic NK cell clones were established from this cell line by limiting dilution. The surface phenotypes of cloned NK cells were IL-2R beta-chain+ CD16+ CD3- IgM-. The clones did not express NK2.1, which is expressed by a half of NK-enriched spleen cells of BALB/c mice. Although the cells contained dense granules reactive to mAb against perforin, they exerted no conventional cytolytic activity against YAC-1. They constitutively expressed Fas ligand (FasL) and specifically killed Fas-positive target cells by fragmenting DNA. This Fas-FasL-mediated killing activity was enhanced by IFN-gamma-inducing factor, a recently identified novel cytokine produced by activated Kupffer cells, but was not affected by other Kupffer cell-produced cytokines, such as IL-12, IL-1beta, and TNF-alpha. Taken together, these findings suggest that hepatic NK cells participate in the immune response as effector cells through the Fas-FasL system in collaboration with cytokines from Kupffer cells.

Human peripheral blood CD4+ and CD8+ T cells express Th1-like cytokine mRNA and proteins following in vitro stimulation with heat-inactivated Brucella abortus

Defining the pattern of lymphokine production associated with Brucella abortus is critical for advancing the development of B. abortus as a vaccine carrier. In the present study we investigated the ability of heat-inactivated B. abortus or lipopolysaccharide from B. abortus to induce lymphokine production from purified human T cells in vitro. Gamma interferon (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-5 induction was assayed by mRNA-specific PCR and by enzyme-linked immunosorbent assay and bioassay for protein production. Following depletion of monocytes and B cells, B. abortus increased IFN-gamma and IL-2 mRNA expression in purified T cells compared with expression in unstimulated cells. In contrast, no IL-5 mRNA expression and only transient low-level IL-4 mRNA expression and no IL-4 protein secretion were detected. Phytohemagglutinin or phorbol myristate acetate plus ionomycin induced mRNA and protein for all these cytokines. Similar results were obtained with LPS purified from B. abortus. Removal of NK cells did not reduce lymphokine production, and enriched NK cells did not express IFN-gamma mRNA or secrete IFN-gamma protein in response to B. abortus, indicating that NK cells were not the responding population. Both CD4+ and CD8+ populations produced IFN-gamma and IL-2 in response to B. abortus. Preincubation of resting T cells with B. abortus or LPS from B. abortus for 7 days induced their differentiation into Th1-like cells as judged by their subsequent lymphokine response to phorbol myristate acetate plus ionomycin. These results suggest that B. abortus can induce differentiation of Th0 into Th1-type cells.

The presence and cytotoxicity of CD16+ CD2− subset from PBL and NK cells in long-term IL-2 cultures enhanced by Prothymosin-α

The aim of this study was to characterize the LAK cell subpopulation on which Prothymosin-α (ProTα) exerts its enhancing effect on cytotoxicity. We investigated the role of ProTα on LAK induction from peripheral blood lymphocytes (PBL) and NK-enriched cells, both cultured for 5 weeks with IL-2 and ProTα. The different cultures were separated into several subsets throughout the culture time using two color fluorescence activated cell sorting (FACS) and CD56, CD 16, CD2 and CD8 monoclonal antibodies. Each cell subset was then tested for cytotoxicity against K562 and Daudi cells. The CD16+ CD2− subset from both, PBL and NK-enriched cells, was the only subset on which ProTα had an effect, significantly enhancing this subpopulation. Within the CD 16 population, the cells CD 16+ CD2+ were the most cytotoxic, although CD 16+ CD2− cells were also cytotoxic. ProTα only potentiated the cytotoxicity of CD 16+ CD2− subset significantly, with 29% and 41 % for K562 and Daudi cells, respectively.

Expression and function of CD7 molecule on human natural killer cells.

The CD7 molecule, one of the earliest T-lymphocyte Ag expressed during ontogeny, has recently been demonstrated to facilitate activation of T cells and to preferentially activate TCR-gamma/delta + subset of T cells. The CD7 Ag is also expressed on human NK cells, but its function has not been determined. In this study, expression and function of CD7 Ag on highly enriched NK cells (94 +/- 3% mean +/- SD, n = 12) obtained by negative selection from peripheral blood of normal donors were investigated. The CD7 Ag was found to be expressed at a significantly (p < 0.002) higher level on fresh NK cells than on IL-2-activated, NK cells. CD7 on human NK cells was found to be a signal-transducing molecule with a rapid increase in cytoplasmic free calcium observed on binding of anti-CD7 mAb to the surface of NK cells. Cross-linking of CD7 induced expression of surface activation molecules such as CD25, CD71, HLA-DR, CD69, and CD54. Activation by anti-CD7 mAb cross-linked to plastic or through goat anti-mouse Ig also induced a variety of NK cell functions: it stimulated secretion of IFN-gamma, led to proliferation of NK cells, as measured by [3H]thymidine incorporation, and significantly enhanced cytotoxicity of NK cells against K562 targets (p < 0.03). However, CD7 on NK cells did not seem to transduce a lytic signal, because it neither mediated redirected killing of Fc gamma R+ murine mastocytoma P815 cells nor triggered lysis of a hybridoma expressing the antibody in a membrane-bound form. CD7 molecules appeared to have a regulatory role in adhesion of NK cells to fibronectin, because cross-linking of CD7 on resting NK cells significantly augmented their adhesion to fibronectin-coated plastic surfaces. However, this induced adhesion was not associated with increased expression of beta 1-integrins on NK cells. Thus, CD7-mediated signals appear to augment function of adhesion molecules on NK cells, which may be involved in NK cell activation by providing both anchorage and costimulatory triggering.

Triggering of the cytotoxic activity of murine natural killer and lymphokine-activated killer cells through the NK2.1 antigen.

The NK2.1 alloantigen, one of the few NK-specific markers in the mouse, identifies a subset rather than all splenic NK cells. On the basis of the cell surface expression of a variety of Ag of the lymphocytic and monocytic lineages, we establish that BALB/c NK2.1+ cells are heterogeneous and that their phenotype is quite similar to the one reported for NK1.1+ cells. A NK2.1- cell population of the same size and related phenotype was also identified in fresh NK-enriched cell suspensions. After stimulation with IL-2, the only phenotypic trait that distinguishes NK2.1+ and NK2.1- cell subsets is precisely the expression of the NK2.1 Ag. To investigate whether NK2.1+ and NK2.1- lymphokine-activated killer cells would also be identical in their cytotoxic activity, NK-enriched spleen cells were cultured for 5 days with IL-2, sorted afterward on the basis of NK2.1 expression, and compared for their capacity to lyse YAC-1 targets. NK2.1+ cells are significantly more lytic than NK2.1- cells, suggesting that the anti-NK2.1 mAb, used for cell sorting, could have triggered NK2.1+ cells and enhanced their lytic activity. In support to this hypothesis we show that 1) immobilized anti-NK2.1 mAb induces granule exocytosis by LAK cells, 2) soluble anti-NK2.1 mAb specifically inhibits NK and LAK cell-mediated lysis of 4LO3311 hybridoma cells secreting anti-NK2.1 mAb, and 3) binding of anti-NK2.1 mAb selectively enhances the lysis of NK-sensitive targets. Furthermore, the successful activation of NK2.1+ cells induced by anti-NK2.1 F(ab')2 or F(ab) mAb fragments indicates that the triggering mechanism is different from reverse antibody-dependent cellular cytotoxicity and does not require cross-linking of the NK2.1 molecule. Our results strongly suggest that the NK2.1 molecule is implicated in a post-binding NK cell signaling event, and point out the possible functional relevance of this NK-specific Ag in non-MHC-restricted cytotoxicity.

The murine NK2.1 antigen: a 130 kD glycoprotein dimer expressed by a natural killer cell subset of the spleen, thymus and lymph nodes

Murine natural killer (NK) cells express a few antigens not found on other leukocyte subsets. The NK1.1 antigen, that is present in only a few mouse strains, has been extensively characterized whereas our knowledge of the NK2.1 antigen, which is more commonly expressed, remains, as yet, limited. Our laboratory has previously reported the production of a mAb (4LO3311) recognizing a murine NK cell-specific molecule with a similar strain distribution as the NK2.1 antigen formerly defined with an NZB anti-BALB/c antiserum. In this study, we demonstrate by sequential immunoprecipitation that 4LO3311 represents the first NK2.1 antigen-specific mAb. This reagent was used to immunoprecipitate the NK2.1 antigen from 125I-labeled lysates of fresh NK-enriched spleen cells. SDS-PAGE analyses revealed that the NK2.1 antigen is expressed at the cell surface as a N-glycosylated disulfide-linked protein dimer with ~65 kD subunits. The NK2.1 antigen is likely to be anchored in the plasma membrane by a peptide moiety since its expression on NK cells was not affected by treatment with phosphatidylinositol-specific phospholipase C. In addition to be present on a splenic NK cell subset, the NK2.1 antigen is shown to be expressed by a small number of CD4 −CD8 −thymocytes and by a subset of CD4 − CD8 −IgG − lymph node cells. Finally, it is shown here that unlike the NKR-P1, the rat homologue of the murine NK1.1 antigen, neither the NK2.1 nor the NK1.1 antigen is expressed by polymorphonuclear leukocytes.

Interleukin 12 is required for the T-lymphocyte-independent induction of interferon gamma by an intracellular parasite and induces resistance in T-cell-deficient hosts.

Immunity against the intracellular protozoan Toxoplasma gondii is highly dependent on interferon gamma (IFN-gamma). We have previously shown that, in addition to T lymphocytes, natural killer (NK) cells can be stimulated by the parasite to produce this cytokine by a reaction requiring adherent accessory cells and tumor necrosis factor alpha. We now demonstrate that a recently characterized cytokine, interleukin 12 (IL-12), is also necessary for parasite-induced IFN-gamma synthesis by NK cells. Anti-IL-12 antibodies completely inhibited T. gondii or bacterial endotoxin-stimulated IFN-gamma production by NK-enriched spleen cells from severe combined immunodeficient mice. Moreover, potent NK cytokine responses were induced by the combination of IL-12 and tumor necrosis factor alpha. In addition, adherent spleen cells from scid/scid mice or thyoglycollate-elicited macrophages from BALB/c animals produced high levels of both IL-12 (p40) and tumor necrosis factor alpha mRNAs when exposed to either live tachyzoites, parasite extracts, or endotoxin, confirming that these cytokines are produced by accessory cells. Finally, in vivo studies showed that treatment with recombinant IL-12 results in prolonged survival of scid mice after infection with T. gondii by means of a response dependent on both IFN-gamma and NK cells. Together the data argue that IL-12 is required for the T-cell-independent triggering of NK cells by intracellular parasites and that the cytokine may be useful for inducing this protective pathway in immunodeficient hosts.

Effects of IL-7 and CLMF on highly enriched NK cells: Bjørn Naume, Maurice Gately∗ and Terje Espevik. Institute of Cancer Research, University of Trondheim, N-7006 Trondheim, Norway and ∗Hoffmann LaRoche Inc., Nutley, New Jersey, USA

Effects of IL-7 and CLMF on highly enriched NK cells: Bjørn Naume, Maurice Gately∗ and Terje Espevik. Institute of Cancer Research, University of Trondheim, N-7006 Trondheim, Norway and ∗Hoffmann LaRoche Inc., Nutley, New Jersey, USA

Activation of CD16+ effector cells by rheumatoid factor complex. Role of natural killer cells in rheumatoid arthritis

The role of natural killer (NK) cells in rheumatoid arthritis (RA) remains unclear. A pathogenetic function of rheumatoid factors (RF) also has not been defined. In the present studies, natural killer (NK) cells were examined as a model for FC gamma receptor type III-positive (FC gamma RIII+) cells, with regard to their interaction with RF. NK cell antigen CD16 (FC gamma RIII) and CD56 expression and functional NK and antibody-dependent cell-mediated cytotoxicity (ADCC) activity were compared in peripheral blood lymphocytes and autologous synovial fluid lymphocytes (SFL) of RA patients. Peripheral blood lymphocytes and SFL showed normal CD56 expression. In contrast, both the frequency and the density of CD16 antigen were decreased in SFL. Furthermore, diminished NK cytotoxicity and a significant decrease in ADCC were observed in SF NK cells. In subsequent in vitro studies with normal fresh NK cells, it was demonstrated that IgG-containing RF complexes from RA patients induced a modulation of FC gamma RIII structure from the NK cell surface, a decrease in NK activity, and a complete loss of ADCC. When purified RF was incubated with NK-enriched cell lines from RA patients, increased transcription and subsequent production of interferon-gamma and tumor necrosis factor alpha were observed. These data suggest a direct involvement of RF complexes in the pathogenetic process of chronic inflammation in RA.

Concomitant Effect of 2′‐Deoxycoformycin on Natural Killer Cell Activity and Tumour Cell Sensitivity to Lysis in Hairy Cell Leukaemia—Discordant Effects of Alpha Interferon

The effects of 2'-deoxycoformycin (dCF) and alpha interferon (IFN-alpha) on natural killer (NK) cell-enriched fractions and hairy cell (HC) targets from three patients with HC leukaemia (HCL) were investigated. There was no significant increase in NK activity when either the HC targets or NK-enriched cells were preincubated with dCF. However, preincubation of both HC and NK cells with dCF resulted in increased NK activity. Culture of enriched NK cells with IFN-alpha enhanced their activity. However, preincubation of HC targets with this drug in the presence or absence of dCF resulted in a protective effect. Maximal NK activity towards HCL was obtained when the target tumour cells were separately precultured with dCF and the NK-enriched effectors precultured with dCF + IFN-alpha. The effect of dCF and IFN-alpha was also measured using the standard K562 cells as targets for NK activity. dCF enhanced NK activity following preculture of both effector and target K562 cells, but IFN-alpha did not reduce K562 cell susceptibility to NK lysis as it did for HC cells. Our findings suggest that (a) dCF and IFN-alpha, which are used to treat HC, could function via activation of NK cells, (b) effects on both effector and tumour target cells should be taken into account, and (c) caution should be exercised in extrapolating the effects of NK-cell activity against K562 cells to those on HC targets.

Mechanism of K562-induced human natural killer cell inactivation using highly enriched effector cells isolated via a new single-step sheep erythrocyte rosette assay

In this study, we used preparations highly enriched in human natural killer (NK) cells to further characterize the mechanism of taget-cell-induced NK inactivation. Highly enriched populations of NK cells were obtained by a newly developed, single-step sheep red blood cell rosette assay. This method, which did not require any incubation steps to facilitate cell contact, permitted a rapid and efficient isolation if NK cells from adherent-cell-depleted peripheral blood lymphocytes. The non-rosetted cells had high NK activity, possessed large granular lymphocyte (LGL) morphology and expressed the NK-associated antigens Leu-11a, Leu-7, OKM1 and NKH-1. In contrast, the rosetted cells had significantly lower NK activity, possessed typical lymphocyte morphology and expressed the T-cell-associated marker OKT3. Next, we examined the ability of these NK-enriched effector cells (EC c) to become inactivated by K562. Functional studies revealed that EC c lost ≥95% of their lytic capacity following incubation with K562 at a ratio of 2/1 for 6 h. However, to achieve this level of inactivation, it was essential that the cell suspension be gently mixed every 90–120 min. Inactivation was not due to cell death and did not reflect changes in the percentages of cells bearing the Leu-11a, Leu-7, OKM1 and NKH-1 antigens, but was associated with an increase in cell surface concentration of OKM1. As judged by gross morphology, the percentages of LGL in EC c before and after treatment with K562 were essentially the same. Finally, K562-treated EC c also lost their ability to mediate antibody-dependent cellular cytotoxicity (ADCC), suggesting that both NK-cell-mediated cytotoxicity and ADCC may involve a common lytic pathway.

LGL-1: a non-polymorphic antigen expressed on a major population of mouse natural killer cells.

Abstract Rat mAb have been raised to mouse liver-derived large granular lymphocytes (LGL). One of these mAb (4D11) binds specifically to mouse LGL and appears to recognize a non-allelic determinant on NK-active cell populations. The Ag recognized by 4D11 is expressed on LGL of all mouse strains tested, including C57BL/6 (B6), BALB/c, C3H/HeJ, and SJL/J; thus, we have provisionally called this Ag LGL-1. Analysis of various lymphoid and hemopoietic tissues has indicated that only normal tissues known to contain NK activity have 4D11+ cells. With B6 and B6 congenic strains, a positive correlation exists between the number of LGL in a sample and the percentage of 4D11 immunofluorescence-positive cells detected by flow cytometric analysis. Dual color immunofluorescence analyses indicate that some LGL-1+ cells are also stained for Ly-1 and Thy-1. A very small subset exists that is weakly positive for CD3 and LGL-1. However, virtually no cells are seen which co-express LGL-1 and Ly-2. LU activity against YAC-1 targets was increased 7- to 700-fold in LGL-1+ spleen cells obtained by cell sorting from several different strains of mice (B6, BALB/c, C3H/HeJ, SJL/J, and athymic/nude). Sorted, LGL-1- spleen cells contained little or no NK activity. Cells positively selected for LGL-1 also contained between 50 and 60% LGL by morphology. By using facilitated in vitro antibody plus C' treatments, the majority of NK activity can be depleted from both B6 spleen and liver-derived leukocyte populations enriched for NK cells. mAb 4D11 was also shown to precipitate a protein of approximately 87 kDa from the surface of enriched murine NK cells. This mAb should prove valuable for understanding the role of NK cells in the immune response.

Poly-N-acetyllactosamine structures on murine cell surface T200 glycoprotein participate in natural killer cell binding to YAC-1 targets.

Cell-surface murine T200 glycoprotein has been implicated in the binding of NK cells to certain susceptible tumor targets. The existence of poly-N-acetyllactosamine structures on T200 glycoprotein and the ability of lactosamine-type oligosaccharides to inhibit NK cell-mediated cytotoxicity suggest that these structures may also be important in NK-target binding. To further identify and characterize these structures, relevant saccharides and reconstituted membrane liposomes containing fractionated effector cell membrane proteins were tested for their ability to block conjugate formation. Under base line conditions, the majority of plastic-non-adherent, Percoll-fractionated, NK-enriched splenocytes that formed conjugates with NK-susceptible YAC-1 targets functioned as lytic effectors in a single-cell cytotoxicity assay. These effectors were blocked in their ability to bind to YAC-1 targets by the addition of N-acetyllactosamine [Gal(beta 1,4)-GlcNAc] and chitobiose [GlcNAc(beta 1,4)GlcNAc], but not by saccharides lacking lactosamine-type linkages. Liposomes prepared from octyl-beta-D-glucopyranoside-extracted YAC-1 and NK-enriched effector cell membranes interfered with conjugate formation, whereas liposomes prepared from NK-insensitive P815 cells were inconsequential. Surface radiolabeled effector cell membrane proteins were fractionated by tomato lectin-Sepharose 4B (poly-N-acetyllactosamine-specific) column chromatography. Tomato lectin-bound material was enriched in a glycoprotein identical with T200, which, when incorporated into liposomes, was a potent inhibitor of effector-target binding. This inhibitory capacity was abrogated by treatment of liposomes with Ly-5 mAb (T200 mAb) or the lactosamine-specific enzyme endo-beta-galactosidase. When T200 was purified by mAb affinity chromatography and incorporated into liposomes, it was a potent inhibitor of conjugate formation, an effect that was blocked by pretreatment of T200-containing liposomes with Ly-5 mAb or endo-beta-galactosidase. These data provide additional evidence that T200 can mediate binding of NK cells to YAC-1 targets, and that poly-N-acetyllactosamine-type structures on NK cell surface T200 glycoprotein are important in the binding process.

Target cell directed NK inactivation. Concomitant loss of NK and antibody-dependent cellular cytotoxicity activities.

We investigated the inactivation of human NK cells, a population of large granular lymphocytes (LGL), with K562, an NK-sensitive target cell (TC) and KLCL, an NK-resistant TC, but which can be lysed by NK cells via antibody (Ab)-dependent cellular cytotoxicity. NK-enriched effector cells (ECc) were first treated with either K562 or Ab-coated KLCL (Ab-KLCL). After incubation, ECc were separated from their TC then examined for residual NK and ADCC activities, phenotypic changes, and changes in LGL morphology. K562-treated ECc and Ab-KLCL-treated ECc, when retested against the inactivating TC, respectively, lost greater than 90% of their lytic activities. However, K562-treated ECc lost 60 to 70% of their activity against Ab-KLCL, whereas Ab-KLCL-treated ECc lost less than 10% of their activity against K562. In contrast to what we observed with K562-treated ECc, we detected significant reductions in plasma membrane expression of Leu-11a and Leu-11b on Ab-KLCL-treated ECc. Although the proportion of OKM1+ cells remained unchanged after the inactivation process, the density of OKM1 on both K562-treated ECc and Ab-KLCL-treated ECc increased significantly. Morphologic analysis revealed no apparent differences in the percentages of LGL before and after treatment with K562 or Ab-KLCL. Finally, IL-2 restored lytic potential to both K562-treated ECc and Ab-KLCL-treated ECc and, in addition, IL-2-induced enhancement of Ab-KLCL-treated ECc was accompanied by a partial reexpression of Leu-11a. These data support the hypothesis that NK-cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity may result from a common lytic mechanism, although the initiation steps and regulation of the pathway are distinct.

Inhibition of natural killer cell activity by eicosapentaenoic acid in vivo and in vitro

To examine the effects of in vivo eicosapentaenoic acid (EPA) on natural killer (NK) cell activity, C3H He mice each received a single intraperitoneal bolus of an emulsion of trieicosapentaenoyl-glycerol (EPA-TG). Spleen cells were tested for NK activity using 51Chromium-release assays against YAC-1 target cells. Forty eight hours after injection, NK activity was inhibited in a dose-dependent manner. EPA-TG emulsion also inhibited the NK activity of NK-enriched effector cells. Decreased cytotoxicity was first noted 24 hr after injection; it resumed the baseline by 7 days. The addition of EPA-TG emulsion to a cytotoxicity assay system resulted in moderate depression of NK activity. These results demonstrate that EPA has significant immunomodulatory effects on NK activity.