Rat natural killer cell and cytotoxic T cell lysis of H-2-negative murine embryonal carcinoma cells

H-2-lacking murine embryonal carcinoma (EC) cells have been proposed as universal targets for natural killer (NK) effectors from different species because their killing appeared to be uncomplicated by potential T cell effector mechanisms (Stern, P. L. et al., Int. J. Cancer 1981. 27:679). While some previous studies had shown that murine cytotoxic T cells were unable to lyse EC cells, rat T killers are shown here to be active against these targets and to be distinguishable from NK cells. Percoll density fractionation of rat peripheral blood lymphocytes enriches in parallel for NK-mediated lysis of both EC or YAC target cells. These NK cells unlike T cells, do not mediate lectin-dependent and cell-mediated cytotoxicity (LDCC) of NK-insensitive target cells. This procedure is thought to reveal the total cytolytic potential of stimulated T cell populations, regardless of specificity. In contrast to previous results with mice, we found that allogeneically primed rat cytotoxic T cells can kill murine EC cells in LDCC and, further, that rat cytotoxic T cells, generated by stimulation with mouse spleen cells in vitro, can lyse murine EC cells directly. This demonstration of T cell lysis of EC cells suggests that either there is a novel mechanism of lysis operating without requirement for major histocompatibility complex (MHC) structures, or EC cells express some hitherto unidentified MHC-like structures on their cell surface.     

In vivo modulation of thymus-derived lymphocytes with monoclonal antibodies in mice. III. Spontaneous and natural cytotoxic effector cells

Cytotoxic effector cell populations in murine spleen can be characterized by the phenotype of the cytotoxic cells or the nature of target cells. Lytic events can be antigen-specific, MHC-restricted and clonal, or target cell-specific but apparently non-MHC-restricted. Two cytotoxic effectors of this latter category are spontaneous and natural killers. Normal spleen cells from (BALB/c X DBA/2J)F1 mice (CDF1) cultured without added antigen develop a population of Thy-1+ spontaneous cytotoxic lymphocytes (SCTL) that lyse the DBA/2J mastocytoma P815, as well as the BALB/c-derived plasmacytomas MOPC-11 and SP2/0. Cold target competition experiments reveal the BALB/c-derived plasmacytomas MOPC-11, SP2/0, J558 and the A strain-derived T cell lymphoma YAC-1, but not normal lymphoblasts, block the lysis of P815 target cells. Thus, while these tumour cells appear to express common antigens which are recognized by SCTL cells, plasmacytomas such as J558 are not susceptible to lysis by SCTL. The relationship of SCTL to natural killer (NK) cells was examined. In-vivo treatment of mice with monoclonal anti-Thy-1 antibody leads to a rapid loss of SCTL and precursors from the spleen, but there is a concomitant increase in NK cell activity.     

Dendritic cells and IFN-alpha-producing cells are two functionally distinct non-B, non-monocytic HLA-DR+ cell subsets in human peripheral blood.

At least two distinct HLA-DR+ cell subsets lacking surface markers specific for B cells, monocytes or other known lineages are present in human peripheral blood. One subset is non-adherent to plastic, produces interferon-alpha (IFN-alpha) when incubated with cytomegalovirus-infected target cells and provides an accessory function required for natural killer (NK) cell-mediated lysis of such cells. These non-adherent HLA-DR+ cells express the surface antigen recognized by antibody anti-D44 and do not stimulate mixed leucocyte reaction (MLR). The other HLA-DR+ cell subset is loosely adherent to plastic, produces only minimal levels of IFN-alpha when incubated with cytomegalovirus-infected target cells and does not provide the accessory function required for NK cell-mediated lysis of such cells. These HLA-DR+ cells stimulate a strong MLR, do not express D44 antigen and meet the criteria of dendritic cells (DC) morphologically and functionally.     

Evolutionary Conservation of a Human Function-Associated Molecule on Murine Natural Killer Cells: Expression and Function

Using a novel anti-natural killer (NK) cell monoclonal antibody (MoAb), we have recently identified an evolutionary conserved function-associated molecule (FAM) present on fish, rat and human NK cells. This molecule is involved in NK cell function as anti-FAM MoAbs inhibit cytotoxicity, stimulate lymphokine secretion and inhibit conjugate formation between effector cells and target cells. We now have examined murine NK cells for the presence of this structure. It was observed by two-colour flow cytometric analysis that the anti-FAM MoAb 5C6 specifically bound to a subpopulation of nylon wool non-adherent splenic lymphocytes (19–20%). The expression of the FAM molecule was restricted to NK cells that expressed the NK1.1 antigen. Neither T cells, B cells, nor macrophages reacted with the anti-FAM MoAb. Analysis of FAM expression in various lymphoid tissues revealed that splenocytes expressed the greatest numbers of MoAb(+) cells. Generation of lymphokine-activated killer (LAK) cells and adherent tymphokine-activated killer (ALAK) cells resulted in higher levels of FAM expression. The anti-FAM MoAb 5C6 also detected the presence of FAM on fresh SCID NK cells. It was demonstrated that the anti-FAM MoAb 5C6 inhibited the lysis of target cells by endogenous NK cells, activated NK cells, 5d LAK cells, ALAK cells and SCID NK cells. Moreover, conjugate assays demonstrated involvement of this molecule in recognition between NK cells and target cells.     

NK and LAK Susceptibility Varies Inversely with Target Cell MHC Class I Antigen Expression in a Rat Epithelial Tumour System

Several cell clones derived from cell lines obtained from a rat thyroid carcinoma, induced by in vivo injection of the Kirsten murine sarcoma virus into thyroid gland, and from its spontaneous lung metastases were analysed for their major histocompatibility complex (MHC) class I antigen expression. The susceptibility to natural killer (NK) cell lysis of these clones, differing in their levels of MHC class I antigen expression, was determined and found to vary inversely with the target cell MHC level, confirming numerous reports of the literature. We then tried to localize the step of the multistage natural cytotoxic process, in which class I antigens could interfere, and tested first whether lymphokine (IL-2) activation of the killer (LAK) cells could overcome the differences in MHC class I expression of target cells. As this did not appear to be the case, we studied the binding step by either a cold target inhibition assay and a target binding assay and found that target cells expressing class I antigens show a lower competitive capacity for effector cells than targets not expressing such antigens, indicating that this interference may occur, at least in our system, in the binding step of the cytotoxic process.     

Production and Characterization of a Novel Monoclonal Antibody Inhibitory for Murine Natural Killer Cell Activity

Natural killer (NK) cells are considered to play an important role in tumor surveillance. The killing of tumor target cells by NK cells is the result of a complex series of sequential binding, signal processing and lytic events. However, the mechanism which NK cells use to recognize tumor targets is poorly understood. To further study the cell-surface molecules involved in tumor recognition, we immunized rats against cloned murine T cells with NK activity (DBA/2.1) and generated rat-mouse hybridomas which were screened for the ability to block lytic activity of DBA/2.1 effector cells. Culture supernatants from one IgM-producing hybridoma, designated S1C4, were found to consistently inhibit DBA/2.1-mediated lysis of YAC-1 target cells. Endogenous splenic NK activity was also diminished in the presence of S1C4 monoclonal antibody (mAb) while alloantigen-specific cytotoxic T lymphocyte (CTL) activity was not affected. S1C4 mAb appears to react with effector cell-surface structures involved in the recognition/adhesion phase of NK activity since pretreatment of effector cells with mAb S1C4 inhibits their ability to bind to YAC-1 target cells. ELISA studies revealed that the S1C4 antigen is expressed by a range of lymphoid cell lines, as well as by DBA/2.1 cells and fresh splenic NK cells. S1C4 mAb were shown to react with 22, 24, 30, and 46 kiloDalton (kDa) DBA/2.1 cell membrane components on immunoblots performed under reducing conditions. These structures do not correspond to any known recognition/adhesion molecules, suggesting that mAb S1C4 defines novel cell membrane components involved in NK cell function.     

Target-Cell Sensitivity to Natural Killer-Cell Lysis is Determined by the Expression of a Novel Antigen in Conjunction with Major Histocompatibility Complex Class-I Molecules

Recently, a panel of monoclonal antibodies (MoAbs) was developed that identified a novel tumour-cell antigen conserved across species (mouse, rat and man). Fluorescence-activated cell sorter (FACS) analysis demonstrated that this antigen was expressed at highest levels on human tumour cell lines sensitive to natural killer (NK)-cell lysis. These MoAbs inhibited NK cell lysis of K562 target cells (by up to 90%), as well as a variety of other NK-sensitive target cells. Biochemical analyses revealed that the MoAbs reacted with a polypeptide of 42 kDaitons, distinct from other known cell surface antigens [1]. Now the expression of this antigen has been analysed further with a panel of 24 tumour cell lines to determine its role in NK cell function. The expression of target cell major histocompatibility complex (MHC) molecules in conjunction with sensitivity to NK cell lysis was examined also. For each of the 24 cell lines it was found that the level of expression of the novel target cell antigen determined the sensitivity of the celt line to NK cell lysis. However, the level of MHC antigen expression could modulate target cell sensitivity to NK cell lysis, in that high levels of MHC class-I molecule expression resulted in a target cell that was insensitive to NK cell lysis regardless of the level of expression of the novel antigen. Thus, for most transformed cell lines, sensitivity to NK cell lysis appeared to be determined by the expression and levels of the novel antigen in association with MHC class-I molecules.     

Rat NKp46 activates natural killer cell cytotoxicity and is associated with FcepsilonRIgamma and CD3zeta

NKp46 has been identified in the human, rat, mouse, monkey, and cattle. We have generated a monoclonal antibody, WEN23, against rat NKp46. By flow cytometry, NKp46 is expressed by all natural killer (NK) cells but not by T cells, B cells, granulocytes, monocytes, dendritic cells, or macrophages. Thus, NKp46/WEN23 is the first NK cell-specific marker in the rat. In a redirected lysis assay, preincubation of the effector cells with WEN23 augmented lysis of the Fc receptor (FcR)+ murine tumor target cells, indicating that NKp46 is an activating NK cell receptor. Moreover, preincubation of the effector cells with WEN23 F(ab')2 fragments reduced killing of target cells, confirming the activating function of NKp46 and indicating that the mouse tumor target cells express a ligand for rat NKp46. Lysis of FcR- mouse and human tumor target cells was reduced after incubation of effector cells with WEN23, suggesting that rat NKp46 recognizes a ligand that is conserved between primates and rodents. By Western blot and immunoprecipitation using WEN23, NKp46 is expressed as a monomer of approximately 47 kDa in interleukin-2-activated NK cells. The immunoreceptor tyrosine-based activation motif bearing adaptor proteins CD3zeta and the gamma chain of FcRI for IgE (FcepsilonRIgamma) with NKp46 from lysates of NK cells, indicating that rat NKp46 activates NK cell cytotoxicity by similar pathways as CD16.     

Laminin inhibits the recognition of tumor target cells by murine natural killer (NK) and natural cytotoxic (NC) lymphocytes.

Tumor cells sensitive to lysis by murine natural killer (NK) or natural cytotoxic (NC) cells were shown to bind laminin. They bound 125I-labeled laminin in a time- and concentration-dependent manner, and binding of the radioactive laminin was inhibited by excess cold laminin. In the presence of laminin, cell-cell aggregation occurred. Murine tumor cells not sensitive to NK/NC-mediated killing bound much less laminin, and laminin did not induce aggregation of these cells. The addition of exogenous laminin to NK or NC cytotoxicity assays reduced target lysis in a dose-related manner. Reduction of lysis was due to an inability of NK/NC cells to bind to the targets. Target cells pretreated with laminin were reduced in their ability to cold-target compete for NK-mediated lysis of untreated target cells. These effects were unique to laminin. The control proteins (fibronectin and thyroglobulin) had no effect on NK activity. Finally, inhibition of cytolytic activity by laminin appeared to be specific for NK/NC cells. Laminin had no effect on cytolysis mediated by alloimmune cytotoxic T lymphocytes regardless of whether the targets did or did not bind laminin.     

A HLA-Cw6 specific single-chain antibody fragment (scFv) recognizing a natural killer cell receptor epitope

Major histocompatibility complex (MHC) class I molecules induce inhibitory signals on natural killer (NK) cells via killer cell immunoglobulin-like receptors (KIR). We recently reported a human single-chain antibody (scFv#1), which recognizes an epitope on HLA-Cw6 (genotype: *0602). Flow cytometry showed scFv#1 binding to HLA-Cw6 (strong) and also to HLA-Cw2, 4, 5 (very weak) but not to HLA-Cw1, 3, 7, 8. The presumptive epitope of the antibody fragment, which includes residues Asn77 and Lys80 was verified by introducing point mutations into HLA-Cw6 encoding cDNAs. Asn77 --> Ser77 (N77S) and Lys80- -> Asn80 (K80N) mutants of Cw6 lost scFv#1 binding capacity whereas an additional mutation at aa position 90 (Asp-->Ala, D90A) did not influence scFv#1 binding characteristics. Since residues 77 and 80 of HLA-C are directly involved in KIR/MHC interaction, we expected the induction of target cell lysis upon addition of scFv#1 when bringing NK and HLA-Cw6 positive cells together. To prove this interference, we performed Cr-release assays, using Cw*0602 and mock-transfected K562 erythroleukemia cells as targets and freshly prepared peripheral blood NK cells as effector cells. scFv#1 appeared to influence KIR on ligand binding and restored lysis at low effector to target (E/T) ratios. Pan HLA class I antibody W6/32 did not show such effects. Taken together scFv#1 binding patterns with mutagenized HLA-Cw6 and Cr-release assays are strong evidence that the scFv#1 epitope on HLA-Cw6 is at or close to the binding site of CD158a.     

Induction of Natural Killer Cell Activity and Allocytotoxicity in Human Peripheral Blood Lymphocytes after Mixed Lymphocyte Culture

The K-562 tumour cell is a highly susceptible target for natural killer (NK) cell lysis by lymphocytes of human peripheral blood. We have studied the antigenic relationship between the recognition sites for lysis of lymphoid and various tumour target cells by cytolytic T lymphocytes (CTL) and NK cells induced in mixed lymphocyte cultures (MLC). The characteristics of these two effector cell types have also been investigated. It was demonstrated that fresh NK cells lose their NK lytic activity when cultured alone. Cell lines not susceptible to lysis by fresh NK cells are lysed by MLC-induced NK cells. There is no antigenic relationship between the recognition sites for the alloreactive T lymphocytes and MLC-generated NK cells expressed on the lymphoid target cells and the tumour target cells, respectively. The MLC-generated alloreactive T cells and NK cells are not identical. The MLC-generated NK cells are different from the fresh NK cells present in the peripheral blood.     

Inhibition of natural killer-cell mediated cytolysis with monoclonal antibodies to restricted and non-restricted epitopes of the leucocyte common antigen.

Three monoclonal antibodies recognizing different epitopes of the leucocyte common molecule, CMRF-11 (against the restricted or B-220 leucocyte common molecule), CMRF-12 and CMRF-26 [each against a different epitope on the non-restricted or T200 leucocyte common (CD45) molecule], were tested for their effects on lymphocyte cytotoxicity. The individual monoclonal antibodies inhibited human natural killer cell-mediated cytolysis (NK-CMC) weakly, but a mixture of CMRF-11 + 12 + 26 antibodies inhibited cytolysis more consistently and to a greater extent. This mixture did not inhibit cytotoxic T lymphocytes derived from secondary mixed lymphocyte cultures. The CMRF-11 + 12 + 26 mix was shown to inhibit a post-conjugate formation stage of lysis at the effector cell level. Inhibition of NK-CMC of a wide range of target cells, including the T-cell lines Jurkat, HSB2 and Molt 4, was demonstrated.     

Characterization of a novel monoclonal antibody against human perforin using transfected cell lines.

The role of perforin in cytotoxicity is controversial. This paper characterizes a novel monoclonal antibody (anti-Phu) against human perforin, using murine cell lines transfected with human perforin cDNA. The antibody specifically stains human perforin in transfected mouse CTLL-2. Anti-Phu blocked granule-mediated haemolysis in an in vitro assay using intact granules isolated from the natural killer (NK)-like human cell line YT, indicating that perforin is a major granule component causing lysis of red blood cells (RBC) in this assay. Inhibition of haemolysis by anti-Phu demonstrated that the antibody binds to undenatured protein as well as fixed perforin molecules. However, the antibody did not inhibit lysis by an allospecific T-cell clone or by YT cells. This could be due to an extremely tight contact between effector and target cell, preventing the antibody from interfering with perforin function by steric hindrance. Physiologically this may reduce lysis of bystander cells. The anti-Phu antibody is a useful tool for further studies of perforin-induced cytotoxicity in vitro and in vivo.     

CD45 molecule cross-linking inhibits natural killer cell-mediated lysis independently of lytic triggering.

The fact that certain CD45 [anti-leucocyte common antigen (LCA)] monoclonal antibodies (mAb) inhibit natural killer (NK) cell non-major histocompatibility complex (MHC)-restricted cytolysis led to the suggestion that these mAb block a 'trigger' for NK cell lytic activity. However, the discovery that the intracytoplasmic portion of the leucocyte common molecule has protein tyrosine phosphatase activity raises the possibility that the mAb initiate a direct inhibitory signal, independent of the triggering apparatus. To clarify this, we have tested the ability of CD45 antibodies to trigger NK cells and redirect cytotoxicity against mAb-producing hybridoma cells and autologous monocytes, an approach which has identified other cytotoxic trigger molecules. Peripheral blood NK cells failed to kill the CD45 antibody-producing hybridomas, although a CD3 antibody expressing hybridoma was susceptible to cytotoxic T-cell lysis. Furthermore, the CD45 mAb CMRF-12 + 26, 13.3 and HuLyM4 did not redirect lysis of autologous monocytes by NK cells, whereas the isotype-matched CD16 mAb did so. Bivalent CD45 antibody was necessary to block NK lysis of K562, as F(ab')2 but not F(ab') fragments of CMRF-12 + 26 antibody inhibited killing. Capping of the LCA appeared to correlate with the ability of the CD45 mAb to block killing, suggesting that cross-linking of LCA molecular isoforms on the NK cell surface is required for CD45 mAb to inhibit non-MHC-restricted cytolysis.     

Inhibition of natural killer cell-mediated cytotoxicity by lipids extracted from Mycobacterium bovis BCG.

Several studies have demonstrated an augmentation of natural killer (NK) cell-mediated cytotoxicity by various adjuvants including BCG. Inhibitory effects of BCG have also been reported, particularly for relatively high doses. Because the cell wall of Mycobacterium bovis BCG contains a high proportion of lipids, the possibility was considered that these lipids may modulate NK activity. A total lipid fraction was extracted from Mycobacterium bovis BCG and used for the lipid modulation of NK effector and target cells. Treatment of effector or target cells resulted in decreased membrane fluidity and decreased NK cell-mediated cytotoxicity in both cases. Pretreatment of target cells did not affect the binding between target and effector cells, as shown in the single cell assay, whereas pretreatment of effectors resulted in inhibition of conjugation. It was further demonstrated that treatment of target cells which were first programmed for lysis protected these cells from subsequent lysis during the killer cell independent lysis stage. The results of this study suggest that adverse effects of BCG treatment on immune functions may be mediated by BCG derived lipids.     

A human lymphocyte-associated antigen involved in cell-mediated lympholysis

Human lymphocyte function antigen (HLFA) is a cell surface protein defined by two monoclonal antibodies MHM23 and MHM24. It is present on both B and T lymphocytes but in greater amounts on the latter. Both antibodies precipitated antigen, from radiolabeled HSB-2 cells, which ran as two chains on sodium dodecyl sulfate polyacrylamide gel electrophoresis at 180 and 94 kDa. Neither antibody inhibited binding of the other, indicating that distinct epitopes were recognized. Both antibodies were shown to inhibit HLA-restricted lysis of influenza virus-infected and Epstein-Barr virus-transformed target cells by cytotoxic T lymphocytes. Blocking occurred at the level of the effector cells and in the presence of subsaturating concentrations of antibody. Both reagents also inhibited lysis of K562 cells, mediated by natural killer cells. These blocking effects differ from the inhibitory effects of monoclonal anti-HLA ABC and anti-suppressor cytotoxic T cell antibodies which inhibit only HLA-restricted lysis when present in saturating amounts. It is concluded therefore that HLFA is likely to be involved in the nonspecific adherence or lytic functions of killer cells rather than specific antigen recognition.     

Trichinella pseudospiralis larvae express natural killer (NK) cell-associated asialo-GM1 antigen and stimulate pulmonary NK activity.

Natural killer (NK) cell function was evaluated in mice infected with either Trichinella pseudospiralis or T. spiralis larvae. T. pseudospiralis-infected mice consistently demonstrated augmented pulmonary NK cell-mediated clearance of YAC-1 tumor cells in vivo but failed to display enhanced splenic NK cell-mediated lysis of the same tumor cells in vitro. Attempts to alter NK cell function in vivo by the injection of anti-asialo-GM1 antibody resulted in anaphylaxis and death of the hosts infected with T. pseudospiralis but not of mice infected with T. spiralis. Immunofluorescence studies demonstrated that larvae of both species bound large quantities of anti-asialo-GM1 antibody but did not react with another NK-associated antibody, anti-NK-1.1. Even though anti-asialo-GM1 antibody reacted with larvae of both species, intravenous injection of antibody elicited anaphylaxis only in hosts that were infected with T. pseudospiralis.     

Kinetic Analysis of Lymphokine-Activated Killer (LAK) Cells: Lytic Parameters and Determination of LAK Cell Frequency

Kinetic analysis was used to define lytic events in murine lymphokine-activated killer (LAK) cell-mediated tumour cell lysis. The maximum rate of target cell lysis (Vmax) and Km (target cell number resulting in 1/2 Vmax) were determined. Single cell lytic assays demonstrated that only LAK effector cells bound to target cells (i.e. non-lytic, bystander lymphocytes did not influence the determination of kinetic parameters) in contrast to natural killer (NK) cell lysis. This finding allowed for LAK cell frequency determinations where Km approximates the concentration of lytic LAK effector cells within a given number of lymphocytes. Frequencies determined in this manner were not significantly different from those obtained using the more cumbersome single cell lytic assay. Furthermore, frequencies determined for the same lymphocyte population against four different NK-resistant tumour targets, that varied in their sensitivity to LAK cell lysis, were not significantly different. In addition, LAK cell lytic programming of target cells was found to be the rate limiting lytic event. This study provides a means of determining reliable estimates of LAK cell frequencies within a lymphocyte population, which will be useful in studies evaluating LAK cytolytic mechanisms and the effects of drugs, biological response modifiers, or disease states on LAK cell lytic activity.     

Tumor cell lysis by T cells distinct from NK cells and alloantigen-specific cytotoxic T cells

The generation and mechanism of tumor cell lysis by cytotoxic T cells derived from natural killer cell (NK) and allospecific cytotoxic T cell (CTL)-depleted precursors were examined. NK cells and the precursors of alloantigen-specific CTL were deleted from human peripheral blood lymphocytes by preincubation with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe). Following phytohemagglutinin activation, CD3(+), CD4(+) or CD8(+), CD11b(-), CD16(-), and NKH1(-) killer cells capable of lysing a broad spectrum of tumor targets were generated. Cytolysis was not strictly lectin dependent as similar killer cells were generated by activating Leu-Leu-OMe-treated T cells with immobilized monoclonal antibodies to the CD3 molecular complex. The rate of tumor cell lysis by these mitogen-activated T cells was slower than that mediated by CD3(-) NK cells. Tumor cell lysis by mitogen-activated killers was inhibited by anti-CD3 but was not restricted by major histocompatibility complex antigen expression on target cells or by CD4/CD8 expression on effectors. Although similar to NK cells in susceptibility to anti-LFA-1 inhibition of killing, these mitogen-activated killer cells were more sensitive to the inhibitory effects of anti-CD2 than were CD3(-)-activated NK-like cells. Thus, tumor cell lysis by CD3(+) cytotoxic cells generated from Leu-Leu-OMe-treated lymphocytes appears to be mediated in part by mechanisms distinct from those employed by CD3(-) NK cells or antigen-specific CTL.