Monoclonal antibodies against the NK cell-FcR and the T3-complex potentiate normal lymphocyte killing

Pretreatment of normal human lymphocytes with monoclonal IgG against the NK cell-FcR (IgG) or the T3 complex was found to potentiate killing of most NK sensitive target cells with the exception of T-cell derived cells. Anti-FcR IgM monoclonals were suppressive for all target cells. IgG anti-FcR mediated potentiation required minute amounts of antibody but was also seen at high anti-FcR concentrations that modulated FcR activity. Potentiated and FcR modulated cells retained anti-FcR IgG on the membrane and conjugated normally to target cells. Anti-FcR potentiation blocked antibody-dependent killing but did not influence lectin-dependent killing, with anti-T3 the opposed effect was seen. Combined anti-FcR and anti-T3 treatment resulted in decreased potentiation. The results suggest that the NK cell-FcR may be activated during normal NK cell killing (without the addition of antibody) as suggested for FcR in B cell triggering.     

Human natural killer cells analyzed by B73.1, a monoclonal antibody blocking Fc receptor functions. II. Studies of B73.1 antibody-antigen interaction on the lymphocyte membrane

In this paper, we characterize the antigen recognized by the monoclonal antibody B73.1 and the modification occurring at the membrane of the positive cells after interaction with the antibody. The B73.1-defined antigen is a protein of 50,000 to 72,000 daltons that is sensitive to pronase but not to trypsin treatment. B73.1 antibody, and its F(ab')2 fragment, directly block, at high concentrations, the binding of IgG antibody-sensitized erythrocytes to the Fc receptors (FcR) of a subpopulation of lymphocytes and neutrophils. B73.1 antibody dissociates rapidly from the positive cells, but concomitant modulation of both B73.1 antigen and FcR is induced when cells are incubated in the continuous presence of antibody or when B73.1 antibody is cross-linked at the cell membrane with an anti-mouse immunoglobulin antiserum. Reaction of lymphocytes with immune complexes also induces modulation of both FcR and B73.1 antigen, without affecting the expression of other antigens on the positive cells. The possibility that the antigen is internalized and digested by the cell after reaction with the antibody is discussed. B73.1 antibody inhibits antibody-dependent cytotoxicity mediated by lymphocytes (K cells) and neutrophils, whereas it does not affect spontaneous cytotoxicity of NK cells. These results suggest the B73.1-defined antigen might be the FcR or a structure closely related to it on K/NK cells.     

The Fc receptor for IgG on human natural killer cells: phenotypic, functional, and comparative studies with monoclonal antibodies

We compare five monoclonal antibodies ( B73 .1, 3G8 , Leu- 11a , Leu- 11b , and VEP13 ) that react with natural killer (NK) cells and polymorphonuclear cells (PMN). We show that all of these antibodies are directed against and inhibit the functional properties of the receptor for the Fc portion of IgG (FcR). Modulation of the FcR on NK cells after reaction with immune complexes induces the disappearance of the antigen(s) recognized by each of the five antibodies. Conversely, the antibodies block binding of IgG-sensitized erythrocytes to the NK cells and PMN and inhibit their ability to mediate cytotoxicity against antibody-sensitized tumor target cells. By using two-color immunofluorescence techniques, we characterize directly the lymphocyte population recognized by these antibodies and show that it is a homogeneous subset that does not bear markers of either B or T cells, with the exception of the 33,000 dalton antigen characteristic of suppressor/cytotoxic T cells present in 20 to 50% of the cells, and the 45,000 dalton receptor for sheep erythrocytes present on 80 to 90% of the cells. The phenotype of the cells reacting with the monoclonal antibodies corresponds to that of NK cells. Cross-competition experiments indicate that these antibodies detect at least two distinct epitopes on FcR, one ( B73 .1) preferentially expressed on NK cells and one or more ( 3G8 /Leu- 11a /Leu- 11b / VEP13 ) preferentially expressed on PMN. The lack of reactivity of these antibodies with B cells suggests that human B cells bear a different FcR from that on NK cells and PMN.     

Expression of markers shared between human natural killer cells and neuroblastoma lines

Summary Neuroblastoma is a tumor of neuroectodermal origin arising most commonly from the adrenal medulla. We have examined the ability of several monoclonal antibodies which recognize markers predominantly expressed on human natural killer (NK) cells to react with neuroblastoma cell lines in vivo derived sections of tumor. HNK-1 (Leu 7) is a monoclonal IgM antibody which recognizes a carbohydrate epitope on NK cells and a wide range of tumor cell types. We have shown that HNK-1 recognizes the human neuroblastoma lines SMS-KCNR, SMS-KAN, NMB/N7, and IMR/5. Expression of this antigen on cell lines can be slightly increased by retinoic acid-induced differentiation of the cells. N901 (NKH1), a monoclonal antibody raised against interleukin 2-dependent human NK cell lines also recognizes all human neuroblastoma cell lines examined. This expression is independent of differentiation induction and levels remain unaltered following retinoic acid treatment of the cell lines. Lastly, with monoclonal antibody 49H.8, it has been found that reactivity of the lines is weak until induction of differentiation, after which highly significant increases of reactivity are seen. 49H.8 recognizes several cryptic carbohydrate antigens with varying affinities, shown to identify mouse and rat NK cells. In contrast to other NK markers, human neuroblastoma cell lines did not express significant reactivity with B73.1, Leu 11b, or Leu 18. Immunohistochemical staining of sections of human neuroblastoma tumors correlated with the in vitro findings; however, staining with N901 and 49H.8 was only seen on frozen sections, not paraffin-embedded. The significance of shared NK cell-neuroblastoma/neuron antigens is currently under investigation.     

Characterization of an antigen expressed by human natural killer cells

A monoclonal antibody, anti-N901, was produced by fusing NS-1 myeloma cells with spleen cells of a mouse immunized with human CML cells. This antibody was reactive with a subpopulation of peripheral blood LGL, including the natural killer cells. Monocytes, granulocytes, B cells, T cells (T3+ cells), erythrocytes, and platelets were nonreactive. The N901-positive cells in the peripheral blood were heterogeneous with respect to expression of other cell surface antigens. The majority of N901+ cells co-expressed T11, Mo1, and HNK-1, whereas a smaller percentage expressed T8. Ia, T3, T4, Mo2, or B1 antigens were very uncommon on N901+ cells. The heterogeneity of the N901+ LGL was further investigated by examining the expression of N901 antigen on a series of cloned normal human NK cell lines. N901 antigen was expressed by each of the NK cell lines tested, and by a minority of cloned T cell lines without NK activity. Anti-N901 does not block NK activity and can be used to rapidly purify functional NK cells for further study.     

Monoclonal antibodies against T cell differentiation antigens initiate stimulation of monocyte/macrophage oxidative metabolism

Within the first minute after incubation with the mouse anti-human T cell orthoclone monoclonal antibodies OKT3, OKT4, and OKT8, and in the absence of complement, human monocytes generate a burst of highly reactive oxygen metabolites as detected by a luminol-dependent photometric chemiluminescence (CL) assay. The kinetics of the CL responses to these antibodies are identical to that induced by OKM1, the monoclonal antibody to human monocytes and granulocytes. With regard to CL response intensities, OKM1 induces the maximal response and those of OKT3, OKT4, and OKT8 closely reflect the proportion of T cell subsets recognized by these antibodies in peripheral blood. This reaction is also observed when monoclonal antibodies against mouse Lyt surface determinants (Lyt-1 and Lyt-2) and Thy-1 antigen are tested against murine spleen cells. This murine model was further used to investigate the specificity and the mechanism of this reaction. It was demonstrated that the CL response is Lyt antigen specific, occurs upon addition of monoclonal IgG but not IgM antibodies, requires the concomitant presence of CL-producing cells (CLPC) (promonocytes, monocytes, macrophages, and/or granulocytes) and of fully differentiated T cells, and lastly, is mediated via a T cell opsonization process. Selective blockade of bone marrow cell Fc receptors (FcR II) with monoclonal anti-mouse FcR II antibody inhibits the CL response to IgG2b anti-T cell antibody-coated thymocytes and thus strongly suggests that the stimulation of CLPC oxidative metabolism in this model results from the binding of opsonized T cells to plasma membrane Fc receptors. These observations lend additional support to increasing evidence that the initiation of effector functions by monoclonal anti-T cell antibodies may be strictly dependent upon the presence of monocytes and/or macrophages.     

Natural killer cells in the blood and bone marrow of the rhesus monkey

Natural killer (NK) cells in peripheral blood lymphocytes (PBL) and bone marrow (BM) cells of the rhesus monkey were detected by their functional activity against K562 cells. Animals could be grouped into "high" or "low" NK responders, a trait found to be consistent over a period of 2 years. NK active cells in PBL were in the nonadherent population, with the majority bearing Fc receptors and a further subdivision of these into CR+ (complement receptor) and CR- NK cells. Of 10 monoclonal antibodies directed against different epitopes of human lymphocytes, OKT11, OKT10, and Leu 11 showed reactivity with rhesus NK cells. Only OKT10 was reactive with the effector site of the cell, as shown by its capability to block NK function. Of the Leu 11 monoclonal antibodies (a, b, c), Leu 11c was nonreactive while Leu 11a and Leu 11b were shown by immunofluorescence to bind to 7 to 21% of PBL; Leu 11b was also cytotoxic to the NK cells. Leu 11b did not prevent binding of Leu 11a to PBL, suggesting reactivity of these antibodies with different epitopes. Percoll fractionation of PBL and BM revealed a greater enrichment of NK activity with BM; also, with PBL peak NK activity occurred in fractions 4 and 5 while this occurred in fraction 5 with BM. Although Percoll PBL fractions contained a higher percentage of Leu 11b cells, the NK activity of the BM fractions was proportionately greater. The majority of PBL cells with NK activity were FcR+ while significant activity could be attributed to FcR- cells of BM, in both the unseparated and Percoll fractions of each tissue. The data suggest NK active cells of BM may be distinct from those found in PBL.     

Functional characterization of LFA-1 antigens in the interaction of human NK clones and target cells

In the present studies we analyzed the role of LFA-1 antigens in the interaction between NK clones and target cells. The use of various cloned NK cell lines allowed us to analyze homogeneous populations of NK cells which ordinarily comprise only a small fraction of peripheral blood lymphocytes and are extremely heterogeneous with respect to phenotype and specificity. Indirect immunofluorescence with monoclonal antibodies against the alpha (MHM24) and beta (MHM23) chains of the LFA-1 antigen revealed similar patterns of positive reactivity with all NK clones. Both monoclonal antibodies exerted a significant blocking effect on NK cytotoxicity against target cells such as Molt-4 and CEM, whereas the inhibition was very weak against other targets such as K562 and HSB cells. Additive blocking effects were seen when both monoclonal antibodies MHM23 and MHM24 were added to the cytotoxicity assays. When we compared the inhibitory effect of MHM23 and MHM24 on uncultured peripheral blood NK cells and IL 2-activated NK cells, inhibition of cytotoxicity also was found to be primarily dependent on the individual target cells. Thus, the inhibitory activity of anti-LFA-1 antibody was shown to be independent of the phenotypic and functional heterogeneity of the NK clones, activated NK cells, and unstimulated NK cells utilized in these studies. These blocking effects were found to be independent of the LFA-1 antigen expression on the target cell membrane and inhibition occurred only when antibody was bound to the effector cells. Comparison of the effects of anti-LFA-1, anti-T3, and anti-clonotypic antibodies against a Ti-like structure of different NK clones with a mature T cell phenotype demonstrated that each of these antibodies acts on the effector cells in an independent and additive fashion. However, unlike T3 and NKTa antigen, LFA-1 antigen expression is not modulated by cell surface interaction with antibodies specific for this molecule.     

Human monocyte-mediated cytotoxicity: the use of Ig-bearing hybridomas as target cells to detect trigger molecules on the monocyte cell surface

We recently reported the preparation and characterization of a monoclonal antibody, 32.2, specific for the high-affinity Fc receptor (FcR) for IgG on human monocytes. We have utilized the hybridoma cell line producing this antibody as a target for monocyte-mediated cytotoxicity. The hybridoma was selected for stable sublines that expressed high quantities of surface 32.2 immunoglobulin (Ig) through flow cytometry. Monocyte-mediated cytotoxicity, with these sublines used as targets, was evaluated with the use of a 51Cr-release assay. It was found that monocytes could efficiently lyse the hybridoma cells (HC 32.2) bearing surface Ig directed to the high-affinity FcR. Consistent with the specificity of the 32.2 antibody for an epitope on the high-affinity receptor outside of the ligand binding site, human IgG did not block monocyte killing of HC 32.2. In contrast, monocytes could not mediate lysis of hybridoma cells bearing high levels of antibody directed to other monocyte cell surface molecules, in particular, class I MHC molecules, the C3bi receptor, and the My 23 antigen. The effect of IFN-gamma on the ability of monocytes to mediate lysis of the 32.2 Ig-bearing hybridomas was also assessed. Monocytes cultured in the absence of IFN-gamma could lyse the hybridoma line expressing high levels of 32.2 Ig as efficiently as monocytes cultured in the presence of IFN-gamma. However, untreated monocytes were less able than IFN-gamma-treated monocytes to kill HC 32.2 expressing lower levels of Ig. Thus, IFN-gamma may enhance the efficiency of monocyte-mediated antibody-dependent killing under conditions where limited antibody is available on the target. These studies demonstrate that the high-affinity FcR on monocytes can act as a cytotoxic trigger molecule for killing of tumor cell targets and that this trigger does not require specific binding to the Fc binding epitope. These results further encourage possible clinical application of the 32.2 monoclonal antibody in tumor therapy.     

Characterization of natural killer cells induced in the peritoneal exudates of mice infected with Listeria monocytogenes: a study of their tumor target specificity and their expression of murine differentiation antigens and human NK-associated antigens

That M1/70, a monoclonal anti-murine macrophage antibody, recognizes murine natural killer cells (NK) and that there is an increase in NK following intraperitoneal infection with live Listeria monocytogenes (LM) was previously reported. Here, LM-induced NK cells were further characterized with respect to tumor target specificity and the expression of murine mast cell, mononuclear phagocyte, and lymphocyte differentiation antigens plus human NK-associated antigens. The M1/70-selected NK (Mac 1 NK) lysed Yac 1, RLmale 1, and WEHI 164.1, but not EL 4 or WTS cells. Immunoprecipitation with M1/70 demonstrated that Mac 1, the antigen recognized by M1/70, was present on NK and thioglycollate-elicited macrophages. Contaminating macrophages in the NK-enriched population did not account for the immunoprecipitated Mac 1. Mac 1 NK that lysed Yac 1 displayed Qa 5, LFA 1, asialo GM 1, Ly 5.1, and NK 1.2, but not Lyt 1, Lyt 2, Mac 2, Mac 3, or Mac 4. Thirty percent of these Mac 1 NK bore Thy 1.2. The presence of Thy 1.2 did not correlate with individual lytic efficiency or cell cycle. Antibodies to human NK antigens Leu 7, Leu 11a, and Leu 15 did not recognize LM-induced NK cells.     

Chemical modification of surfactant protein A alters high affinity binding to rat alveolar type II cells and regulation of phospholipid secretion

Alveolar type II cells express a high affinity receptor for pulmonary surfactant protein A (SP-A), and the interaction of SP-A with these cells leads to inhibition of surfactant lipid secretion. We have investigated the binding of native and modified forms of SP-A to isolated rat alveolar type II cells. Native and deglycosylated forms of SP-A readily competed with 125I-SP-A for cell surface binding. Alkylation of SP-A with excess iodoacetamide yielded forms of SP-A that did not inhibit surfactant lipid secretion and did not compete with 125I-SP-A for cell surface binding. Reductive methylation of SP-A with H2CO and NaCNBH3 yielded forms of SP-A with markedly reduced receptor binding activity that also exhibited significantly reduced capacity to inhibit lipid secretion. Modification of SP-A with cyclohexanedione reversibly altered cell surface binding and the activity of SP-A as an inhibitor of lipid secretion. Two monoclonal antibodies that block the function of SP-A as an inhibitor of lipid secretion completely prevented the high affinity binding of SP-A to type II cells. A monoclonal antibody that recognizes epitopes on SP-A but failed to block the inhibition of secretion also failed to completely attenuate high affinity binding to the receptor. Concanavalin A inhibits phospholipid secretion of type II cells by a mechanism that is reversed in the presence of excess alpha-methylmannoside. Concanavalin A did not block the high affinity binding of 125I-SP-A to the receptor. Neither the high affinity binding nor the inhibitor activity of SP-A was prevented by the presence of mannose or alpha-methylmannoside. The SP-A derived from humans with alveolar proteinosis is a potent inhibitor of surfactant lipid secretion but failed to completely displace 125I-SP-A binding from type II cells. From these data we conclude that: 1) cell surface binding activity of rat SP-A is directly related to its capacity to inhibit surfactant lipid secretion; 2) monoclonal antibodies directed against SP-A can be used to map binding domains for the receptor; 3) the lectin activity of SP-A against mannose ligands does not appear to be essential for cell surface binding; 4) concanavalin A does not compete with SP-A for receptor binding; and 5) the human SP-A derived from individuals with alveolar proteinosis exhibits different binding characteristics from rat SP-A.     

Functional and biochemical analysis of CD16 antigen on natural killer cells and granulocytes

CD16 Ag is associated with the low affinity FcR for IgG expressed on human NK cells and granulocytes. In this study, we demonstrate that NK cells specifically lyse murine anti-CD16 hybridoma cell lines, but do not lyse hybridomas against other cell surface differentiation Ag expressed on NK cells. Moreover, the CD18 structure is involved in the CD16-specific xenogeneic interaction between human effector cells and murine hybridoma target cells. Although interaction with anti-CD16 hybridomas or antibodies triggers the cytolytic mechanism of NK cells, this interaction does not induce cellular proliferation. In contrast to NK cells, CD16+ granulocytes do not lyse anti-CD16 hybridoma cell targets and do not mediate ADCC against antibody-coated human tumor cell targets. These findings indicate a fundamental difference in the antibody-dependent cellular cytotoxicity mechanisms of NK cells and granulocytes. Comparative biochemical analysis of CD16 on NK cells and granulocytes revealed significant differences in the size of the polypeptides obtained after removal of N-linked carbohydrate residues with endo-F and N-glycanase digestion.     

Characteristics of a monoclonal antibody (WT-31) that recognizes a common epitope on the human T cell receptor for antigen

We describe a monoclonal antibody, WT-31, that reacted with all human T lymphocytes. Electrophoretic analysis of the material reacting with WT-31 revealed that it precipitated predominantly an 80-kD disulfide-linked heterodimer from the cell surface-labeled T leukemic cell line HPB-ALL. This heterodimer was identical to the one precipitated with a recently described monoclonal reagent, T40/25, which recognizes a clonotypic structure on HPB-ALL. The target antigen of WT-31 comodulated with T3 after incubation of T cells with excess anti-T3 antibody, indicating that the WT-31 target antigen is associated with T3. We also found that anti-T3 reagents, but not the clonotypic reagent T40/25, blocked binding of FITC-labeled WT-31 to HPB-ALL cells. This indicates that the T cell receptor epitope recognized by WT-31 is located close to the epitopes recognized by the anti-T3 reagents anti-Leu-4 and SPV-T3b but distal from the clonotypic T40/25 epitope. Functional studies showed that WT-31 reacts similar to anti-T3 antibodies. It is mitogenic for resting T cells, blocks cytolysis mediated by alloantigen-specific CTL clones, and induces antigen-nonspecific cytolysis by CTL clones against Daudi target cells. WT-31 did not inhibit the formation of conjugates, but it blocked cytolysis just before or during the Ca2++-dependent programming for lysis. We conclude that WT-31 is an antibody that recognizes a common determinant on the T cell receptor for antigen. The present results support the notion that the two chains of the T cell receptor (alpha and beta) form a functional protein ensemble with the three invariable T3 polypeptide chains (T3-gamma-, delta-, epsilon).     

Selective inhibition of natural killer activity by the monoclonal antibodies OKT10 and VEP10 at the single cell level

The monoclonal antibodies, VEP10 and OKT10, which have been shown to recognize determinants on human natural killer (NK) cells, inhibit large granular lymphocyte (LGL) NK activity against K562, MOLT4, and CEM tumor target cells in the single cell conjugate agarose assay. Inhibition of NK activity by monoclonal antibodies was expressed independently of effector-target cell binding, as inhibitory activity could be demonstrated when the monoclonal antibodies VEP10 and OKT10 were added to preformed conjugates or to the LGLs and targets prior to the binding event. In addition, this inhibition was exerted on the effector cell and not the target cell since VEP10 and OKT10 did not react with determinants on K562 target cells. Furthermore, the 4F2 monoclonal antibody, which reacted with determinants on the LGL and all of the targets used, effected no inhibition of NK activity. Inhibition of killing by OKT10 and VEP10 was specific to endogenous NK activity since the same antibodies did not inhibit antibody-dependent cellular cytotoxicity (ADCC), mixed lymphocyte-generated NK, or cytotoxic T lymphocyte (CTL) activities.     

Induction of calcium flux and enhancement of cytolytic activity in natural killer cells by cross-linking of the sheep erythrocyte binding protein (CD2) and the Fc-receptor (CD16)

Binding of the anti-cluster of differentiation (CD) 2 monoclonal antibody 9-1 causes an increase in the concentration of cytoplasmic-free calcium ([Ca2+]i) in cultured CD3-/CD16+ natural killer (NK) cells. This response did not occur in cultured CD3+/CD16- cytotoxic T lymphocytes (CTL). Anti-CD16 antibodies could partially block the calcium response when NK cells were stimulated with intact antibody 9-1, and antigen-binding fragment F(ab')2 of antibody 9-1 did not produce a calcium response. Thus an interaction of the 9-1 antibody with CD16 Fc receptors was required for the functional effect. The dual interaction of antibody 9-1 with both CD2 and CD16 was demonstrated by comodulation experiments. The cytolytic activity of cultured NK cells was increased by antibody 9-1 but not by F(ab')2 fragments of antibody 9-1. The enhanced lytic activity was blocked by anti-CD16 antibody, anti-CD18 antibody, and anti-CD2 antibodies that do not block the binding of antibody 9-1. This pattern was distinct from antibody-dependent cell-mediated cytotoxicity which was blocked only by the anti-CD16 antibody. Thus antibody 9-1 enhanced cytotoxicity by activating effector cells. There was no enhancement of lytic activity when F(ab')2 of antibody 9-1 were cross-linked with a polyclonal antiglobulin, even though [Ca2+]i was increased. These results show that induction of a [Ca2+]i response is not sufficient to enhance lytic activity in NK cells, and suggest that signals delivered through CD16 are necessary.     

A novel function of the herpes simplex virus type 1 Fc receptor: participation in bipolar bridging of antiviral immunoglobulin G

We describe a novel function of the Fc receptor of herpes simplex virus type 1 (HSV-1), its ability to participate in antibody bipolar bridging. This refers to the binding of a single immunoglobulin G (IgG) molecule by its Fab end to its antigenic target and by its Fc end to an Fc receptor (FcR). We demonstrate that various immune IgG antibodies, including polyclonal rabbit antibodies to HSV-1 glycoproteins gC1 and gD1 and monoclonal human antibody to gD1 blocked rosetting of IgG-coated erythrocytes at IgG concentrations 100- to 2,000-fold lower than required for rosette inhibition with nonimmune IgG. Steric hindrance did not account for the observed differences between immune and nonimmune IgG since rabbit anti-gC1 F(ab')2 fragments did not block rosetting. Murine anti-gC1 or anti-gD1 IgG, a species of IgG incapable of binding by its Fc end to the HSV-1 FcR, also did not block rosetting. When cells were infected with a gC1-deficient mutant, anti-gC1 IgG inhibited rosetting to the same extent as nonimmune IgG. This indicates that binding by the Fab end of the IgG molecule was required for maximum inhibition of rosetting. Bipolar bridging was shown to occur even when small concentrations of immune IgG were present in physiologic concentrations of nonimmune IgG. The biologic relevance of antibody bipolar bridging was evaluated by comparing antibody- and complement-dependent virus neutralization of an FcR-negative mutant and its parent HSV-1 strain. By engaging the Fc end of antiviral IgG, the parent strain resisted neutralization mediated by the classical complement pathway. These observations provide insight into the role of the HSV-1 FcR in pathogenesis and may help explain the function of FcR detected on other microorganisms.     

A triggering structure recognized by G7 monoclonal antibody on porcine lymphocytes and granulocytes.

Monoclonal antibody (mAb) G7 has been developed and appears to recognize a triggering structure on porcine natural killer (NK) cells and granulocytes. G7 mAb binds to approximately 13% of lymphocytes, 70% of monocytes, and greater than 95% of granulocytes. G7 mAb does not react with B cells. G7 mAb immunoprecipitates a heterodispersed molecule of approximately 40 kDa. Functionally, whole but not F(ab')2 fragments of G7 mAb enhance NK killing of Fc receptor positive K562, U937, and MOLT-4 targets but not Fc receptor negative CEM, WEHI-164, or YAC-1 targets. Both whole and F(ab')2 fragments of G7 mAb inhibit lymphocyte-mediated antibody-dependent cellular cytotoxicity. Interestingly, G7 mAb induces dramatic levels of granulocyte killing against nucleated K562 targets. These results suggest that G7 mAb recognizes a trigger molecule involved in porcine cellular cytotoxicity.     

Monoclonal antibodies to Fc receptors for IgG on human mononuclear phagocytes. Antibody characterization and induction of superoxide production in a monocyte cell line

We have utilized monoclonal antibodies against the two IgG Fc receptors (p40 and p72) of U937 cells to stimulate the release of superoxide. The monoclonal antibody (mAb) specific for p40 (IV3) has been described elsewhere. A murine IgG1 mAb specific for the high affinity p72 Fc receptor (designated mAb FcR32 or simply mAb 32) bound to the same p72 precipitated by Sepharose-human IgG as shown by preclearing experiments and by identical isoelectric focussing patterns. Binding of mAb 32 to p72 was independent of the Fc region of the antibody since Fab' fragments of mAb 32 affinity adsorbed p72. The binding of both mAb 32 and human IgG1 to the intact U937 cell was not reciprocally inhibitory, indicating that mAb 32 does not interfere with the ligand binding site of p72. mAb 32 bound to human monocytes, U937, and HL60 cells, but not to granulocytes or lymphocytes. U937 cells cultured in gamma-interferon and 1,25-dihydroxycholecalciferol generated superoxide when incubated with mAb 32 or IV3 followed by cross-linking with F(ab')2 anti-murine Ig. Incubation with mAb 32 or IV3 alone or with 3 of 5 other anti-U937 mAbs cross-linked with anti-murine Ig did not result in superoxide generation. Immune complex-mediated superoxide production was inhibited 80% by IgG, but not by mAb 32 or IV3.     

T11/CD2 activation of cloned human natural killer cells results in increased conjugate formation and exocytosis of cytolytic granules

The T11 (CD2) antigen has been found to be an alternate pathway for antigen-independent activation of resting T cells. T11 triggering also results in activation of NK cells and enhancement of their cytolytic function. The present studies were carried out to further define the mechanisms whereby cytotoxicity is enhanced after T11 activation. A series of clonal human NK cell lines were analyzed after incubation with monoclonal anti-T112 and anti-T113 antibodies specific for different epitopes of the CD2 protein. Anti-T112/3 triggering resulted in increased cytotoxicity against a variety of target cells. Similar results were obtained with F(ab')2 fragments of anti-T112/3, indicating that this effect was not mediated through binding of FcR. The induction of cytotoxicity was found to be associated with increased formation of effector cell-target cell conjugates and with release of secretory granule-localized 35S-labeled proteoglycans. Both enhanced conjugate formation and cytotoxicity could be blocked by anti-lymphocyte function-associated antigen (LFA-1) mAb. Ultrastructural analysis of NK cells after T11 activation demonstrated increased adherence of effector cells to targets and other NK cells as well as a directional reorientation of cytoplasm and intracellular granules toward the area of contact between cells. Discharge of granules occurred into pockets bounded by closely apposed plasma membranes. In the presence of anti-LFA-1 and anti-T112/3, the close apposition and formation of pockets between effector cells and target cells did not occur but the cells exocytosed their intracellular granules. T11 activation of NK cloned cells also resulted in the formation of the homotypic conjugates and autocytotoxicity. As seen with resistant allogeneic targets, autocytotoxicity was mediated by F(ab')2 fragments of T112/3 antibodies and could be blocked by anti-LFA-1 antibody. Ultrastructural analysis of NK cloned cells after T11 activation confirmed the presence of homotypic conjugates with reorientation of effector cells toward one another and discharge of cytolytic granules into pockets formed between NK cloned cells. Taken together, these results indicate that T11-induced cytolytic function of NK cells is, in part, mediated through increased binding of effector cells and targets and that enhanced conjugate formation is at least in part mediated by the LFA-1 antigen. In addition, T11 activation results in the triggering of the cytolytic mechanism of NK cells and the exocytosis of cytolytic granules and their constituents.     

A new monoclonal antibody recognizing an antigen of human lymphocytes similar or identical to Tac antigen

A new mouse monoclonal antibody (HIEI, IgG1 type) that reacts with a cell surface glycoprotein of human lymphocytes was isolated. Membrane immunofluorescence assay showed that HIEI, like the anti-Tac monoclonal antibody, reacted preferentially with activated normal human T-cells and adult T-cell leukemia (ATL) virus (ATLV)-carrying human T- and B-cell lines. However, an interesting difference between HIEI and anti-Tac antibody was that HIEI did not react with ATLV-transformed simian cell lines or those cultured with interleukin-2 (IL-2), whereas the anti-Tac antibody did. The immunoprecipitation assay showed that both HIEI and anti-Tac antibody precipitated a glycoprotein with a molecular weight of 60,000 daltons (gp60) from activated normal T-cells and ATLV-positive T- and B-cells, and also gp53 from MT-2 and MT-2-related T-cell lines transformed with ATLV in vitro by the MT-2 cocultivation method. HIEI inhibited the IL-2-dependent proliferation of normal T-cells, but its inhibitory effect was much weaker than that of the anti-Tac antibody. The anti-Tac antibody interfered with the binding of HIEI to target cells, but HIEI did not block binding of the anti-Tac antibody to the cells. These observations indicate that HIEI antibody recognizes a new antigenic determinant of the human Tac antigen.