Elevated NK-mediated lysis of Raji and Daudi cells carrying fixed iC3b fragments

Raji and Daudi cells were opsonized with C3b, iC3b, and C3d fragments by using purified complement components. The sensitivity of C3-opsonized cells to lysis mediated by low density blood lymphocytes was studied. Raji and Daudi cells carrying C3b or C3d fragments were lysed with similar efficiencies as the nonopsonized cells. The presence of iC3b on the target surface imposed elevated NK sensitivity. The iC3b-mediated enhancement of NK lysis was inhibited when iC3b fragments or rabbit anti-human C3 antibodies were included into the lytic assays. These results indicate that the iC3b fragments fixed on the targets bind to the CR3 on the lymphocytes. Results obtained in immobilized conjugate-lytic assays showed that iC3b-opsonized targets interact more readily with the lymphocytes. This was reflected by the elevated proportion of lymphocytes that were bound to the iC3b-carrying targets. The proportions of conjugates in which target damage occurred were similar with the control and with the iC3b-carrying cells. It seems therefore that opsonization of targets with iC3b leads to recruitment of effector lymphocytes due to contact with their CR3. However, once the effector-target contact is established, the triggering of lytic function does not seem to be influenced by the iC3b/CR3 bridge.     

Studies on human blood lymphocytes with iC3b (type 3) complement receptors. I. Granular, Fc-IgG receptor positive and negative subsets in healthy subjects and patients with systemic lupus erythematosus

By using the OKM1 monoclonal antibody and the fluorescence-activated cell sorter to identify lymphocytes bearing iC3b (type 3) complement receptors, two principal populations of OKM1+ lymphocytes have been identified in human peripheral blood. One subset exhibited azurophilic granules and Fc receptors for IgG stained by Leu-11. The other population did not display FcR, but was enriched in cells reacting with OKT3 and OKT8 (low intensity). In healthy subjects, approximately 60% of CR3+ lymphocytes were granular FcR-bearing cells and only 18% co-expressed OKT3 determinants. In patients with systemic lupus erythematosus (SLE), CR3+ lymphocytes were predominantly FcR negative cells and 71% lacked granules. Only 33% reacted with Leu-11, but 50% co-expressed OKT3, 44% reacted with OKT8+, and 15% were OKT4+. We tested the hypothesis that agranular OKT3+ Leu-11- lymphocytes, such as those found in SLE patients, contained the precursors of natural killer (NK) cells. Leu-11+ cells were removed from normal lymphocytes by complement lysis, and the remaining cells were treated with recombinant IFN-alpha, IFN-gamma, or IL 2. These procedures were ineffective in generating typical NK effector cells. Our studies do not support the hypothesis that CR3+ Leu-11- lymphocytes are the precursors of granular Leu-11+ NK cells.     

A large subpopulation of lymphocytes with T helper phenotype (Leu-3/T4+) exhibits the property of binding to NK cell targets and granular lymphocyte morphology

A discrete subpopulation of lymphocytes sharing several phenotypic characteristics with natural killer (NK) cells was identified within the circulating pool of human lymphocytes that bear the T helper marker Leu-3. This Leu-3+ subpopulation of cells formed cell conjugates with the NK target cell lines K562 and MOLT-4, but did not bind to mouse myeloma and hybridoma cell lines that are insensitive to NK cells. The Leu-3+ lymphocytes binding to NK cell targets contained cytoplasmic granules similar in ultrastructure and cytochemistry to those previously defined in granular lymphocytes with NK function, except that the granules in Leu-3+ cells were smaller and fewer in number. Unlike classical NK cells, however, the granular Leu-3+ cells did not kill the target cells to which they bound, even after treatment with interferon. The proportion of granular Leu-3+ cells with the capacity to bind to NK cell targets was approximately 7% at birth and increased to approximately 21% of the Leu-3+ cells in adults. These observations suggest the possibility of a lineal relationship between the granular Leu-3+ cells and granular Leu-3- cells with NK capability.     

Potentiation of NK cytotoxicity by antibody-C3b/iC3b heteroconjugates

The interaction of two Burkitt lymphoma lines, Raji and Rael, with human C and NK cells was analyzed. Raji cells activate the alternative C pathway (ACP) and then bind C3 fragments. Consequently, the cells become more sensitive to lysis by CR3-bearing NK cells but not to C lysis. In contrast, Rael cells are poor ACP activators, do not bind C3 fragments, and are therefore resistant to C-dependent NK lysis. As suggested earlier, the difference between Raji and Rael could be attributed to the presence or absence of CR2, respectively, on their surface. To potentiate C- and NK-dependent lysis of target cells, we generated heteroconjugates composed of a murine antitransferrin receptor mAb and of human C C3b or iC3b. Antibody-C3b conjugates induced C3 deposition on Rael cells and elevated C3 deposition on Raji cells in human serum. Both Raji and Rael cells coated with antibody-C3b conjugates were efficiently lyzed by the cytolytic ACP in human serum. This conjugate had a small enhancing effect on target cell lysis by NK cells which could be markedly increased by combined treatment of the target cell with antibody-C3b conjugate and C5-depleted human serum. On the other hand, antibody-iC3b conjugates efficiently potentiated lysis of target cells by NK cells in the absence of serum. The iC3b-directed cytotoxicity was mediated by CR3-bearing NK effector cells. Anti-C3 but not anti-mouse Ig antibodies abrogated the activity of the antibody-iC3b conjugate. These results further demonstrate that NK cytotoxicity may be potentiated by opsonizing the target cells with C3 fragments and suggest that antibody-C3b/iC3b conjugates could be potent tools for targeting and potentiation of the lytic action of both C and NK cells against tumor cells.     

Elevated NK sensitivity of Raji cells carrying acceptor-bound C3 fragments

The majority of cell lines derived from Burkitt lymphomas carry CR2 on their plasma membrane cell lines of haematopoietic origin can activate C3 present in human serum through the alternative pathway. However, only the lines that carry CR2 were shown to bind C3 fragments. This bond can be either fixation to acceptor sites or attachment to the CR. Our studies with Raji cells showed that when the possibility for the covalent acceptor bond was eliminated by using methylamine (MA)- or zymosan-treated serum, considerably lower amounts of C3 were bound. In the zymosan-treated serum C3 fragments are present that can bind to receptors but their capacity for acceptor bond is absent. These results indicate that when Raji cell are incubated in human serum some of the generated C3 fragments are bound to acceptors and a lower proportion through the specific interaction with complement receptors. Pretreatment of the CR2 carrying cell lines with human serum elevated their sensitivity to the lytic effect of human blood lymphocytes. We showed in this work that MA-treated serum did not induce this elevation. Zymosan-treated serum under conditions that excluded activation of the residual native C3 molecules, i.e., in the presence of EDTA, did not have the enhancing effect either. These results suggest that the increased lytic efficiency imposed by human serum was due to cleavage of C3 molecules by Raji and fixation of the C3 fragments by acceptor sites. Natural killer cells carry CR3; therefore it is likely that the attached C3 fragments bind also to the effector cells. The C3 molecules could elevate thereby the avidity between the target and the lytic lymphocytes. The observation that C3 fragments are not bound to the surface of CR2 negative lines in spite of their capacity to activate C3 suggests that the receptor molecule is either involved in the activation and/or serves also as an acceptor.     

Spontaneous and lymphokine-induced cytotoxic activity of monkey intestinal mucosal lymphocytes

We determined the capacity of primate (macaque monkey) intestinal mucosal lymphocytes to mediate natural killer cytotoxicity, and characterized the nature of cells mediating this form of cytotoxicity in the intestine. Isolated macaque monkey intestinal mucosal lymphocytes were found to have intermediate levels of spontaneous cytotoxicity against K562 target cells compared to higher values found in lymphocytes of peripheral blood (PBL) and spleen and low values for mesenteric lymph node (MLN) lymphocytes. Intestinal lymphocytes were similar to PBL in having the same range of target cell specificites, in having augmentation of activity by interferon or interleukin 2, and in demonstrating specificity in cold target inhibition studies. Both intestinal and PBL spontaneous cytotoxic function in primates was mediated predominantly by cells bearing antigens cross-reactive with the anti-human monoclonal antibody Leu-11. The percentage of Leu-11+ lymphocytes was significantly lower in isolated intestinal, spleen, and MLN lymphocytes compared to peripheral blood. Furthermore, isolated intestinal lymphocytes differed from PBL in that intestinal Leu-11+ were predominantly Leu-15-, while Leu-11+ PBL were predominantly Leu-15+. These studies demonstrate that the lower spontaneous cytotoxic function of intestinal mucosal lymphocytes compared to PBL is associated with a lower number of effector cells and with effector cells which differ qualitatively in expression of the Leu-15 antigen.     

Lectin-dependent and anti-CD3 induced cytotoxicity are preferentially mediated by peripheral blood cytotoxic T lymphocytes expressing Leu-7 antigen

Monoclonal antibodies against the CD3 antigen and certain lectins can induce interleukin 2 dependent antigen-specific T cell clones to mediate non-antigen specific cytotoxicity. On the basis of this observation, we predicted that it may be possible to identify cytotoxic T lymphocytes (CTL) in peripheral blood without knowing the antigen specificity of these in vivo primed CTL. By using this strategy, peripheral blood lymphocytes were separated into low and high-density fractions on Percoll gradients and were tested for cytotoxic activity in the presence or absence of concanavalin A (Con A) or anti-Leu-4 antibody. Lectin-dependent cellular cytotoxicity (LDCC) and anti-CD3 induced cytotoxicity against both natural killer (NK)-insensitive and NK-sensitive targets were exclusively mediated by low-density CD3+ T lymphocytes. Additional studies indicated that low-density CD3+ T lymphocytes co-expressing Leu-7 antigen preferentially mediated this activity, although in some individuals, significant activity was also observed in the low-density T cells lacking Leu-7. In contrast, high-density CD3+ T lymphocytes and CD16+ (Leu-11+) NK cells (both Leu-7 and Leu-7+) did not mediate nonantigen-specific cytotoxicity under these conditions. The finding that NK cell-mediated cytotoxicity was unaffected by these lectins refutes the hypothesis that lectin-dependent cellular cytotoxicity is simply a result of effector and target agglutination. T cell-mediated cytotoxicity was both lectin and antibody specific. Phytohemagglutinin, Con A, and pokeweed mitogen induced cytolytic activity in the Leu-7+ T cells, whereas wheat germ agglutinin did not. Of the antibodies against T cell-associated differentiation antigens (anti-Leu-2,3,4, and 5), only anti-Leu-4 induced cytotoxicity. This anti-CD3-induced cytotoxicity was essentially completely inhibited by the presence of anti-LFA-1 or anti-CD2 monoclonal antibodies, implicating these molecules in the triggering process. A proportion of the CD3+, Leu-7+ CTL expressed HLA-DR antigens, indicating possible in vivo activation. Because previous clinical studies have indicated that lymphocytes with this phenotype may be elevated in clinical situations associated with immunosuppression and chronic viral infection, this unique subset of CD3+ T lymphocytes may represent a population of in vivo primed CTL possibly against viral antigens.     

Bacterial activation of human natural killer cells. Characteristics of the activation process and identification of the effector cell

We showed previously that contact of human peripheral blood lymphocytes with glutaraldehyde-fixed Salmonella bacteria augmented their cytotoxic capacity against NK-sensitive targets. We have now analyzed the characteristics of the activation and also identified the subsets of lymphocytes responding to bacterial contact. Blocking of protein synthesis with cyclohexamide totally abrogated bacterial induction of activated killing (AK), whereas inhibition of DNA synthesis with mitomycin C did not significantly affect the capacity of lymphocytes to respond to bacterial contact. Both the induction and the effector phase of AK were radioresistant. The AK cells exhibited efficient lytic activity, comparable to that induced by recombinant IL 2 (rIL 2), against NK-resistant targets (including both hematopoietic and solid tumor cell lines). All inducible cytotoxic activity was contained within the subset of lymphocytes expressing Leu-19 (NKH-1) antigen. Leu-19- lymphocytes exhibited no significant NK activity and could not be further stimulated by bacterial contact, rIL 2, or IFN-alpha. Within the Leu-19+ lymphocyte subset, two distinct cell types were present; CD3-, Leu-19+ NK cells and CD3+. Leu-19+ T cells. The CD3+, Leu-19+, T cells mediated low levels of non-MHC-restricted cytotoxicity against K562, but did not respond to bacterial contact, even though rIL 2 could augment their lytic activity slightly. However, the cytotoxic activity of CD3-, Leu-19+ NK cells was significantly augmented by bacterial contact. Within the CD3-, Leu-19+ NK cell population both CD16+ and CD16- cells responded to bacterial activation. The CD3-, CD16-, Leu-19+ cells constituted 1 to 4% of the Percoll-fractionated low buoyant density lymphocytes and accounted for the activation seen within the CD16- lymphocyte population. Thus bacterial stimulation of NK activity seems to be mediated for the most part via CD16+, Leu-19+ cells, and a minor overall contribution is mediated via CD3-, CD16-, Leu-19+ cells. No apparent involvement of T cells was seen in the lytic response of lymphocytes to bacterial contact.     

Lymphocytes stimulated by allogeneic B cell lines cleave the third component of complement and fix C3 fragments. Their nonspecific lytic capacity is elevated against complement receptor type 2-carrying targets

Human blood lymphocytes stimulated in mixed cultures by allogeneic B cell lines were shown to cleave C3 molecules. The B cell lines were derived from Burkitt lymphoma patients: 1) established from their EBV-negative lymphoma, 2) the EBV-positive sublines converted in vitro, and 3) lymphoblastoid cell lines (LCL) i.e., B lymphocytes transformed in vitro by EBV. These cell lines differed considerably in their capacity to stimulate allogeneic lymphocytes. The split products of C3 were detected in the supernatants and on the surface of the activated lymphocytes at levels which correlated with the strength of stimulation. Lymphocytes cultured with LCL had the highest levels of thymidine incorporation blast transformation, C3 cleavage, and C3 fragment fixation. Lymphocytes exposed to the EBV-negative Burkitt lymphomas were stimulated weakly and their C3-activating capacity was low. Irrespective of the efficiency of lymphocyte stimulation induced in the cultures, 60 to 70% of the blasts were found to fix C3 fragments. The majority of the lymphocytes which fixed C3 fragments were T blasts that carried the CD3 marker and expressed IL-2R (CD25). CD4 and CD8 cells were represented with equal frequency in the C3-fragment fixing and C3-fragment negative populations. Pre-exposure of the MLC-activated lymphocytes to human serum increased their cytotoxic capacity toward CR type 2-carrying targets. The enhanced lysis was abrogated by F(ab)2 rabbit anti-human C3d or rabbit anti-CR type 2 antibodies. The results suggest that the C3 fragments fixed on the lymphocytes bind to CR on the targets and elevate the avidity of binding between the two interacting cells. This was also indicated by an increase in the frequency of lymphocyte-target conjugates.     

C3 receptors on human lymphocyte subsets and recruitment of ADCC effector cells by C3 fragments

The presence of C3 receptors on human peripheral blood lymphocytes (PBL) and on the ADCC-exhibiting subset (K cells) thereof was analyzed by rosetting with bovine erythrocytes (Eb) or chicken erythrocytes (Ec) carrying human C3b, C3bi, or C3d. The indicator cells were coated with 20,000 to 100,000 C3 fragments, obtained by C3 activation with purified proteins of the alternative pathway and trypsin treatment. ADCC was studied at the cellular level by means of a plaque assay, with complement-free or complement-carrying indicator cells as targets. Of the total lymphocytes, 12 to 14% bound EC3b; 6 to 8%, EC3bi; and approximately 2%, EC3d. Surface marker analysis indicated that approximately 75% of the C3b-binding lymphocytes in PBL were either B or null cells and approximately 60% of the C3bi-binding cells were T cells, as characterized by the monoclonal antibodies OKT3 and OKT4 or by presence of receptors for Helix pomatia hemagglutinin. Of the K cells, which constituted from 5 to 10% of the total lymphocytes, approximately 20% bound C3b; 30 to 35%, C3bi; and 7 to 8%, C3d. Here the majority of the C3b binders were null cells, and the majority of the C3bi and C3d binders were T cells. Only one-third of the C3b-binding K cells and one-fifth of the C3bi-binding K cells bound both fragments. The nature of these double binding cells is unknown. In contrast, all C3d-binding K cells bound C3bi as well. C3 fragment-carrying target cells did not induce K cell-mediated lysis in the absence of anti-target antibodies but strongly enhanced ADCC in the presence of sublytic concentrations of such antibodies. The rank order for C3 fragment-induced enhancement was C3bi greater than C3d greater than C3b. It reflected the relative proportions of effector cells binding the different fragments. Enhancement was the expression of effector cell recruitment rather than of increased cytolytic activity of individual K cells. This recruitment was selective in that C3b-carrying target cells primarily recruited effector cells of null type, binding C3b, while C3bi- or C3d-carrying targets primarily recruited C3bi and/or C3d-binding K cells of T gamma type. Thus, these experiments show directly at the effector cell level that cell-bound C3 fragments constitute important recognition structures, which strongly amplify ADCC both by recruiting the proper effector cells into the cytolytic reaction and by very significantly decreasing the antibody concentration needed for its induction.     

Augmentation by anti-T3 antibody of the lymphokine-activated killer cell-mediated cytotoxicity

This study showed that a mAb (145-2C11) against the T3 epsilon-chain of the TCR complex augmented the cytotoxic activity of the lymphokine-activated killer (LAK) effectors. The LAK cells were induced by culturing normal spleen cells with purified human rIL-2. Adding alpha T3 at the effector phase of the cytotoxic reactions augmented the LAK-mediated cytotoxicity. The alpha T3-augmented LAK killing was seen only with tumor targets, and there was no increase of killing against Con A-induced lymphoblasts. The augmentation effect was dose dependent on both the amounts of alpha T3 and the number of LAK cells added. A very low concentration of alpha T3 (1/10,000 dilution of culture supernatants) was sufficient to induce alpha T3-augmented LAK-mediated cytotoxicity. Human rIL-2 at 10 to 30 U/ml was sufficient to generate LAK cells for maximal alpha T3 augmentation, whereas 300 to 1000 U/ml of IL-2 were needed to generate maximal LAK activity when tested in the absence of alpha T3. LAK cells generated for longer periods of time showed a progressive increase of alpha T3-augmented cytotoxicity. For some targets, the alpha T3-augmented LAK killing was FcR dependent as evidenced by the ability of alpha FcR mAb 2.4G2 to inhibit, and for others it was not inhibited. The alpha T3-augmented killing did not correlate with the FcR expression on target cells as defined by 2.4G2. The LAK cells were both Lyt-2+ and Lyt-2-, but the LAK cells involved in alpha T3-augmented killing were exclusively Lyt-2+. Preincubation of LAK cells with alpha T3, but not preincubation of targets with alpha T3, resulted in augmented killing suggesting that the alpha T3 effect was unrelated to an antibody-dependent cell-mediated cytotoxicity. Our findings indicate that alpha T3 is a potent reagent to augment the cytotoxic reaction of LAK cells. These results suggested that a relationship might exist between the T3 complex and the cytotoxic activity of a subpopulation of Lyt-2+ LAK cells.     

Mode of in vitro augmentation of natural killer cell activity by recombinant human interleukin 2: a comparative study of Leu-11+ and Leu-11- cell populations in cord blood and adult peripheral blood

Human newborn natural killer (NK) cell activity against K562 target cells was observed to be low compared with adult controls. Although Leu-7 (HNK-1)+ cells were negligible in cord blood, the proportions of Leu-11+ cells were equal to those of adult peripheral blood. Leu-11+ cells sorted from cord blood lymphocytes, as well as from adult lymphocytes exhibited the morphology of granular lymphocytes. In this study, we have investigated the phenotypic characterization of recombinant interleukin 2 (rIL 2)-induced cytotoxic lymphocytes against K562 cells by using anti-Leu-11 monoclonal antibody. Spontaneous cytotoxicity of lymphocytes was restricted to Leu-11+ cells in cord blood, as well as in adult blood, but this activity was low in cord blood Leu-11+ cells as compared with that of adult ones. NK cell activity of adult Leu-11+ cells could not be additionally enhanced after an 18-hr incubation with rIL 2(25 U/ml), whereas rIL 2 could potentiate the cytotoxicity of cord blood Leu-11+ cells approximately to the adult levels. It should be noted that cytotoxic activity of both Leu-11- cells from cord blood and adult blood that had no basal NK cells activity could be significantly potentiated by rIL 2. On the other hand, lymphokine-activated killer cells cytotoxic for HL-60 cell line could not be generated, and no proliferation of the lymphocytes was detected after an 18-hr incubation with rIL 2. It was shown that rIL 2 could not enhance the ability to bind to target cells in Leu-11+ and Leu-11- cells by means of a single cell conjugate assay, but the rate of target lysis of Leu-11+ cells from cord blood was significantly enhanced by rIL 2. These results suggested that rIL 2-induced cytotoxic effector cells were heterogeneous, and rIL 2 might potentiate the cytotoxicity of functionally immature NK cells or NK precursor cells.     

Human T lymphocytes expressing the C3b/C4b complement receptor type one (CR1, CD35) belong to Fc gamma receptor-positive CD4-positive T cells

The phenotypic characteristics of human T lymphocytes expressing the C3b/C4b complement receptor type one (CR1, CD35) were investigated using dual-color surface immunofluorescence and cytofluorometric analysis of stained peripheral blood mononuclear cells (PBMC) from normal individuals. Two to ten percent of PBMC coexpressed CR1 and the CD5, CD2, or CD3 antigen. CR1 was detected on a subset of CD4+ T lymphocytes but not on CD8+ or on Leu-7+ lymphocytes. Costaining for CR1 and for the CD4 subpopulation markers anti-Leu-8, TQ1, OKT17, 2H4, and 4B4 indicated that CR1 on lymphocytes may be coexpressed with any of these phenotypic determinants. All CR1+ lymphocytes expressed Fc gamma receptors (Fc gamma Rs) as assessed by their ability to bind biotinylated dimeric human IgG. The expression of CR1 was increased in mixed lymphocyte reaction with kinetics similar to those of HLA-DR antigen expression. Coexpression of CR1 and Fc gamma R+ may provide a subset of CD4+ lymphocytes with an enhanced ability to bind and respond to C3-bearing complexes of IgG and antigen.     

Signal transduction during human natural killer cell activation: inositol phosphate generation and regulation by cyclic AMP

NK cells mediate both direct cytotoxicity against a variety of tumor cells and indirect (FcR-dependent) cytotoxicity against antibody-coated targets. When cloned human NK cells (CD16+/CD3-) were exposed to NK-sensitive targets for 30 min, the level of inositol phosphates rose two to five times above background. The rise in inositol phosphates induced by NK-sensitive targets was paralleled by an increase in intracellular free calcium concentration ([Ca2+]i). A panel of tumor cells that were resistant to NK cell lysis did not stimulate significant levels of inositol phosphate production and did not induce an elevation of intracellular free calcium. Ligation of the FcR (CD16) with the mAb 3G8 also triggered phosphoinositide turnover. Kinetic experiments demonstrated that stimulation by either susceptible target cells or by FcR ligation led to rapid (less than 1 min) generation of the Ca2+-mobilizing second messenger, inositol trisphosphate, with slower accumulation of inositol bisphosphate and inositol monophosphate. Previous studies have demonstrated that activation of the cAMP-dependent second messenger pathway strongly inhibits NK cell-mediated cytotoxic functions. Treatment of NK effector cells with forskolin to elevate intracellular cAMP levels resulted in a concentration-dependent inhibition of phosphoinositide hydrolysis induced by both NK-sensitive targets and 3G8-mediated FcR ligation. These results suggest that phosphoinositide turnover represents a critical early event in the human NK cell cytolytic process. Moreover, the potent inhibitory effect of cAMP on NK cell cytotoxicity may be explained by the uncoupling of NK receptors from phospholipase C-mediated phosphoinositide hydrolysis.     

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.     

Demonstration of NK cell-mediated lysis of varicella-zoster virus (VZV)-infected cells: characterization of the effector cells

Infection with varicella-zoster virus (VZV) rendered RAJI cells more susceptible to lysis by non-adherent blood lymphocytes. At an effector to target ratio of 80:1 the mean percentage of 51Cr release of VZV-infected RAJI cells was 41 +/- 12%, whereas that of uninfected RAJI cells was 15 +/- 6%. The increased susceptibility to lysis was associated with increased effector to target conjugate formation in immunofluorescence binding assays. The effector cells cytotoxic for VZV-infected RAJI cells were predominantly Leu-11a+ Leu-4- granular lymphocytes as demonstrated by fluorescence-activated cell sorting. The effector cell active against VZV-infected RAJI cells appeared similar to those active against herpes simplex virus (HSV)-infected cells, because in cold target competition experiments the lysis of 51Cr-labeled VZV-infected RAJI cells was efficiently inhibited by either unlabeled VZV-infected RAJI cells (mean 71% inhibition, 2:1 ratio unlabeled to labeled target) or HSV-infected RAJI cells (mean 69% inhibition) but not by uninfected RAJI cells (mean 10% inhibition). In contrast, competition experiments revealed donor heterogeneity in the overlap between effector cells for VZV- or HSV-infected RAJI vs K-562 cells.     

Studies of complement receptors on cytotoxic effector cells in human peripheral blood

The question of whether cells bearing complement receptors (CR) mediate cytotoxicity in vitro against allogeneic Chang liver cell targets was investigated by assessing peripheral blood mononuclear cells (PBMC) from normal humans for cell surface characteristics and cytotoxic capacity before and after depletion of CR+ cells capable of forming rosettes with sheep erythrocytes coated with 19S antibody and mouse complement (EAC) and depletion of Fc receptor-bearing cells capable of forming rosettes with human O+ erythrocytes coated with Ripley antibody (EA-Ripley). PBMC depleted of CR+ cells by density centrifugation contained markedly reduced proportions of phagocytes and sIg + cells and increased proportions of both sIg ?, FcR+ cells as well as cells forming rosettes with sheep erythrocytes (E). PBMC depleted of CR+ cells mediated cytotoxicity to an extent equal to or greater than that mediated by unfractionated PBMC in assays of spontaneous cell-mediated cytotoxicity (SCMC), antibody-dependent cellular cytotoxicity (ADCC), and mitogen-induced cellular cytotoxicity (MICC). Cells harvested from the EAC-rosette enriched pellet mediated cytotoxicity 5- to 10-fold less than unfractionated PBMC; however, the cytotoxic activity of the pellet could not be attributed to CR + effector cells since similar cytotoxic activity was present in cell pellets obtained by density centrifugation of PBMC which had been incubated with E coated with 19S antibody or E alone. PBMC depleted of EA-Ripley rosette-forming cells contained decreased proportions of sIg?, FcR+ cells and increased proportions of CR+ cells; PBMC so depleted contained virtually no SCMC and ADCC effector cell activity. These findings indicate that at least the majority of effector cells which mediate SCMC, ADCC, and MICC do not bear CR.     

Virus-induced enhancement of lymphocyte-mediated antibody-dependent cytotoxicity (ADCC) in vitro

The effect of Parotis virus on antibody-dependent cellular cytotoxicity in vitro (ADCC) of human lymphocytes was investigated in a 51Cr-release assay and, at the effector cell level, in an ADCC plaque assay. Target cells were bovine or chicken erythrocytes, which are not susceptible to natural cytotoxicity (NK) of human lymphocytes. They were not killed when incubated with virus-treated lymphocytes in the absence of antibodies. Treatment of the lymphocytes or the target cells with small amounts of virus, however, resulted in a very significant enhancement of ADCC. The same results were obtained with live or UV-inactivated virus, suggesting that enhancement was a passive phenomenon not requiring infection. Enhancement was already significant after 3 hr of incubation, indicating that it was independent of endogenously released interferon. Enhancement of ADCC by virus was due to effector cell recruitment rather than due to the increase of the cytotoxic potential of the individual K cell. The highest frequency of effector cells was present in Percoll fractions enriched in large granular lymphocytes (LGL). Virus treatment resulted in recruitment of effector cells carrying T cell markers such as the T3 antigen (OKT3+), receptors for sheep erythrocytes, or Fc receptors for IgM. In contrast, the absolute number of K cells carrying the HNK-1 marker (Leu-7) or receptors for C3 fragments was not changed by the virus. It is concluded that Parotis virus enhances ADCC by improving effector cell-target cell contacts, resulting in recruitment of effector cells with T cell characteristics. Recruitment is accompanied by a significant reduction of the antibody concentration needed for ADCC induction. This virus-mediated enhancement of ADCC may be of importance for protection of the host in the early phases of a virus infection in which the amounts of anti-viral IgG antibodies capable of inducing cellular cytotoxicity may yet be very small.     

Lymphocytes infiltrating human ovarian tumors. I. Role of Leu-19 (NKH1)-positive recombinant IL-2-activated cultures of lymphocytes infiltrating human ovarian tumors

Tumor-infiltrating lymphocytes (TIL) were obtained from human ovarian tumors, expanded in the presence of IL-2 in culture and studied for cytotoxicity against fresh autologous and allogeneic ovarian carcinoma (CA) targets. TIL from ovarian tumors grew well in long term cultures, achieving from 8- to 682-fold expansion. TIL cultured with IL-2 were cytotoxic against both autologous and allogeneic fresh ovarian CA targets, and no specificity for autologous tumor could be demonstrated in any of the cultures. In all fresh TIL preparations, CD3+ lymphocytes were the major cell type and contained a high proportion (up to 51%) of activated (IL-2R+) cells as determined by two-color flow cytometry. Sorting of bulk TIL cultures followed by cytotoxicity assays identified the Leu-19+ cells, both CD3+ and CD3-, as effectors of cytotoxicity against autologous and allogeneic tumor cell targets. Cold target inhibition assays showed that allogeneic targets (both ovarian CA and a sarcoma) competed effectively with autologous ovarian CA targets for Leu-19+ effectors in TIL cultures. mAb to Leu-19 or Leu-2a did not block lysis of autologous targets by sorted effectors. OKT3 antibody augmented lysis of autologous targets by CD3+Leu-19- effectors only. These results show that non-MHC-restricted Leu-19+ effectors in cultures of TIL with 1000 U/ml of rIL-2 mediate lysis of autologous and allogeneic tumor cells. The CD3+Leu-19- cells, the main population in these cultures, do not mediate tumor lysis. To determine the phenotype of antitumor effectors in IL-2 cultures of TIL, cell sorting followed by functional assays are necessary.     

Mechanism of cell-mediated cytotoxicity at the single cell level. VI. Direct assessment of the cytotoxic potential of human peripheral blood non-lytic effector-target cell conjugates

Single cell cytotoxicity assays reveal that a large percentage of lymphocytes are unable to kill attached targets in a 4- to 18-hr assay. Additional signals (in the form of lectin or anti-target antibody) delivered to target-bound lymphocytes enable these previously non-lytic lymphocytes to kill attached target cells. This finding was obtained by using a modification of the single cell assay, in which lectin or target cell antibody is incorporated into agarose with preformed lymphocyte-target conjugates. Human peripheral blood lymphocytes (PBL) or Percoll density gradient-enriched large granular lymphocytes (LGL) were used as effector cells in natural killer (NK), antibody-dependent cellular cytotoxicity (LDCC) assay systems. The targets used were NK-sensitive K562 and Molt-4 and NK-insensitive Raji. Several findings were made in the modified single cell assay, namely a) the frequency of cytotoxic NK or ADCC effector cells was not augmented, suggesting that the initial trigger was sufficient for lytic expression in these instances. Furthermore, these results showed that the NK-sensitive targets used do not bind nonspecifically to the LDCC effector cells. K562 coated with Con A, however, serve as LDCC targets. b) The frequency of two target conjugate lysis by NK/K effectors was not augmented by Con A. These results suggest that Con A does not potentiate the killing of multiple targets bound to a single cytotoxic lymphocyte. c) Although conjugates formed between LGL or PBL and NK-insensitive Raji are non-lethal, significant lysis was observed when these conjugates were suspended in Con A or antibody agarose. These results demonstrate that Raji bind to cytotoxic NK, K, and LDCC effector cells, but are lysed only when the appropriate trigger is provided. d) The cytotoxic potential of non-lytic conjugates appears to lie within the low density Percoll fraction, although the high density lymphocytes are able to nonlethally bind to targets. Altogether the results demonstrate that target recognition and/or binding by the effector cells is a distinct event from the trigger or lytic process. The implications of these findings are discussed.