Model for population distributions of lymphocyte-target cell conjugates

A quantitative model for the population distributions of the different types of conjugates formed between cytotoxic T lymphocytes and target cells has been developed. The comparison of the theoretical predictions with data of the literature reveals that the transit populations among the different types of conjugates depends on the lymphocyte-to-target ratio, R, and two constants, k and k1. These constants (where k greater than k1) govern, respectively, the transit populations among conjugates of the type LTi (LTn----LTn-1----...LT), and among LjT conjugates (LT----L2T----...----LmT). We have found that high ratios are necessary to obtain conjugates where multiple T lymphocytes are bound to one target cell, and that under these conditions the predominant conjugate, LjT, varies according to j = 1 + k1R. Conversely, for low values of R the predominant population is of the type LTi, where i also shows a linear dependence on R. Our model explains also why the conjugate LT is normally the predominant population under the experimental conditions reported in the literature. A discussion of the influence exerted by the population distributions of lymphocyte-target cell conjugates on the kinetic of the lytic process for these kinds of effector-target systems has also been made.     

Effect of conjugate size on the kinetics of cell-mediated cytotoxicity at the population level

We developed a model for the kinetics of target cell lysis by cytotoxic T-lymphocytes which accounts for most facts observed at the population level. In contrast to previous models, the following facts: conjugate frequency of cytotoxic T-lymphocytes bound to target cell, dependence of this frequency on the lymphocyte-to-target ratio (R), variation of R with time as target cells are destroyed, and population distributions of the different types of conjugates formed between lymphocytes and target cells, which are involved in the kinetics of these kinds of effector-target systems have been contemplated in the model. The relationship with effector-kinetic analogy models for the lytic process has been discussed. Predictions of the model have been explored and compared with experimental observations about target cell lysis reported in the literature.     

Relationship between target cell recognition and temporal fluctuations in intracellular Ca2+ of human NK cells

Temporal changes in intracellular Ca2+ concentration, [Ca2+]i, of resting human peripheral blood NK cells in response to target cell binding were evaluated by flow cytometry. [Ca2+]i was significantly elevated in PBL and purified NK cells bound to NK-sensitive K562 and HSB2 target cells, but not in those bound to NK-resistant MD1 B-lymphoblastoid cells. Thus, a) the ability of a target cell to elicit a Ca2+ flux response correlated with its sensitivity to lysis of NK cells, and b) adhesion alone was not a sufficient stimulus for response induction. Conjugates of NK cells bound to K562 target cells were sorted onto agarose-coated slides on the basis of relative NK cell [Ca2+]i and evaluated in 19-hr single cell agarose cytotoxicity assays. In contrast to those with basal levels of [Ca2+]i, NK cells with elevated [Ca2+]i bound more strongly to target cells, as judged by the stability of conjugates to sort-related shear forces (p less than 0.01), and more frequently killed the target cell to which they were attached (p less than 0.05). Temporal fluctuations in [Ca2+]i were observed in target-bound NK cells in both the presence and absence of extracellular Ca2+. Thus, influx of extracellular Ca2+ and release of Ca2+ from internal stores both appeared to contribute to the NK cell Ca2+ flux response triggered by adhesion to appropriate target cells. These results support the hypothesis that such fluctuations in NK cell [Ca2+]i constitute an early signal flagging the occurrence of NK cell recognition.     

Natural killer activity against cultured human neural tumor and fetal brain cells

Ten human neural tumor lines and three established from normal human brain were analyzed for sensitivities to natural killer (NK) cytolysis. Compared to MOLT-4, fetal brain cells were sensitive, but those from adult brain and eight of ten neural tumor cell lines demonstrated marked NK resistance. The frequencies of target-binding cells (TBC) and single-cell lysis of glioma cells bound within tumor cell conjugates demonstrated that the resistance of two lines was explained either by a decrease in the frequencies of TBC or reduced ability of bound NK cells to lyse the tumor cell conjugates. A third resistant line demonstrated decreases in both TBC and tumor cell conjugate lysis. Two glioma lines with less NK resistance had greater frequencies of TBC or conjugate lysis than the resistant lines. Thus, NK resistance can result from decreased recognition of targets, diminished NK lysis of bound targets, or a combination of both.     

Characterization of effector-target conjugates for cloned human natural killer and human lymphokine activated killer cells by flow cytometry.

The present studies demonstrate that the intracellular fluorochromes calcein and hydroethidine can be used for quantification of effector-target conjugates involving cloned human natural killer (NK) or interleukin-2 (IL-2) activated human lymphokine activated killer (LAK) cells by dual color flow cytometry without potential artifacts that might result from extensive modification of effector and/or target cell membranes. Cloned NK cells and LAK cells form conjugates with cultured cell lines regardless of susceptibility to lysis. The strength of the interactions in these conjugates was investigated using a variable speed vortexer. Even relatively gentle vortexing disrupted most conjugates involving fresh human peripheral blood lymphocytes (PBL) but only about one-fourth of conjugates between K-562 cells and human PBL that had been cultured with or without IL-2 by this treatment. The rate of conjugate formation for LAK cells was determined to be about 3 times faster than for cloned NK cells, and both rates are considerably faster than the reported rate of formation of cytotoxic T lymphocyte (CTL) target conjugates. The differences in the rate of conjugate formation are apparently not related to target cell specificity, since LAK cells form conjugates with susceptible and resistant cell lines at comparable rates. When effector-target conjugates are incubated at 37 degrees C in the absence of calcium--thereby precluding lysis--the percentage of conjugated LAK or cloned NK cells decreases logarithmically with time. These results suggest that an initial equilibrium between free and conjugated lymphocytes gradually shifts in favor of unconjugated 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.     

Inhibition of human antibody-dependent cellular cytotoxicity, cell-mediated cytotoxicity, and natural killing by a xenogeneic antiserum prepared against "activated" alloimmune human lymphocytes

Xenogeneic antiserum (RH1) was prepared in Lewis rats by hyperimmunization with concanavalin A- (Con A) activated alloimmune human lymphocytes. The antiserum RH1 effectively inhibited human antibody-dependent cellular cytotoxicity (ADCC), cell-mediated cytotoxicity (CMC), and natural killing (NK) in the absence of complement (C). Inhibition by RH1 was dependent on the dilution of antiserum employed and the number of cytotoxic lymphocytes present during cytolysis. Pretreatment of lymphocytes with RH1 or the presence of RH1 in culture did not inhibit lymphocyte proliferation stimulated by Con A, phytohemagglutinin, or allogeneic cells; lymphokine production as measured by leukocyte-inhibiting factor production; antibody-dependent C lysis; or CMC mediated by murine cytotoxic T lymphocytes. Analysis of the mechanism of inhibition of cytotoxicity by RH1 revealed that 1) RH1 was not cytotoxic for human lymphocytes at 37 degrees C in the absence of C; 2) purified F(ab')2 fragments were equally inhibitory as whole serum; 3) pretreatment of lymphocytes with RH1 effectively inhibited their capacity to mediate ADCC, CMC, or NK, and this effect was reversible by culturing the cells overnight at 37 degrees C; 4) RH1 did not inhibit target cell binding by K cells, effector cells of ADCC, or alloimmune T cells, but did inhibit binding by NK cells; and finally, 5) the addition of RH1 to preformed lymphocyte-target conjugates in a single cell cytotoxicity assay inhibited killing of the bound target cells in all three systems without disrupting the conjugates. Collectively, these findings suggest that RH1 antiserum interacts with structures present on the surfaces of cytotoxic lymphocytes that are involved in the activation of the lytic mechanism(s) or with the actual lytic molecule or molecules themselves. Furthermore, the ability of RH1 to inhibit ADCC, CMC, and NK during the post-binding cytolytic phase of these reactions indicates that binding and cytolysis are distinct and separate events in all types of cell-mediated cytolysis.     

Evidence of a dynamic role of the target cell membrane during the early stages of the natural killer cell lethal hit

The role of membrane movement during the stages of human NK cytolysis has been examined by using the bifunctional protein cross-linking reagent, glutaraldehyde. The binding stage was inhibited when K562 target cells or NK cells were pretreated with glutaraldehyde. When added post-binding, after initiation of calcium pulse, glutaraldehyde did not dissociate conjugates, but inhibited NK cytolysis. In contrast to the early stages of NK cytolysis, glutaraldehyde enhanced lysis during the terminal stage, killer cell independent lysis ( KCIL ). Lysis of the preprogrammed target cells, however, was enhanced only when glutaraldehyde was added immediately after dispersion of the conjugates, before target cell lysis. The mechanism of enhancement of lysis during the terminal stages of cytolysis was further explored in assays for NK cell-derived cytolytic factor (NKCF). L929 target cells prebound with NKCF were lysed more readily in the presence of glutaraldehyde, but as in KCIL , maximum enhancement of lysis occurred when glutaraldehyde was added immediately after NKCF was bound to the target cell. These results suggest that the target cell membrane may play a dynamic role during the terminal stages of the NK lethal hit.     

Quantitation and sorting of vitally stained natural killer cell-target cell conjugates by dual beam flow cytometry

We have detected formation of stable associations, or conjugates, between fluorescein diacetate-(FDA) stained human natural killer (NK) cells and Hoechst 33342-(HO342) stained tumor cells by dual laser flow cytometry. Conjugates in mixtures of effectors and targets emitted both green (FDA) and blue (HO342) fluorescence. This was confirmed by cell sorting. More than 90% of the conjugates included one target and one effector cell. Conjugate formation frequency was temperature independent between 4 and 37 degrees C, optimized by 10 min, and stable for 1 hr. Enrichment of effector populations for cells mediating lysis of standard NK targets and for cells reacting with OKM1, Leu-7, and Leu-11b monoclonal antibodies also enriched conjugate-forming cells. Lysis of either OKM1+, Leu-11b+ effector subpopulations with antibody and complement eliminated, but treatment with these antibodies alone had no effect on, conjugate formation. Effector pretreatment with Leu-4 or 3A1 and complement increased the frequency of conjugation slightly. Flow-determined frequencies of NK-conjugate formation with 14 target cell lines correlated well with data derived from standard microscopic assays. However, the flow method was more rapid, could be used when target and effector were of comparable size, and permitted isolation of conjugates by sorting.     

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.     

Enhanced lysis of herpes simplex virus type 1-infected mouse cell lines by NC and NK effectors

Spontaneously cytotoxic murine lymphocytes lysed certain cell types infected by herpes simplex virus type 1 (HSV-1) better than uninfected cells. The levels of virus-directed lysis varied widely from target to target, and we found that differences in virus-directed lytic efficiency could be attributed both to the characteristics of HSV-1 replication in the different targets and to the subgroup of natural effector cells which mediated lysis. Although HSV-1 adsorbed to the surface of all the target cells, those in which the virus replicated more efficiently were lysed to a greater extent. As targets, we used cell lines that, when uninfected, were spontaneously lysed by NK cells (YAC-1) or by NC cells (WEHI-164). We also used a fibroblastoid cell line (M50) and a monocytic tumor line (PU51R), which were not spontaneously killed. Using complement-mediated elimination of Qa-5-positive or asialo-GM1-positive NK cells to distinguish NK from NC activity, we found that NK cells lysed HSV-1-infected YAC cells better than uninfected cells, and an NC-like activity selectively lysed HSV-1-infected WEHI cells. In addition, we showed that both NK and NC cytotoxicities contributed to the lysis against the HSV-1-infected fibroblastoid line, M50, but the infected PU51R cells were killed by only NK effectors. These findings were consistent with the results of experiments performed to define the role of interferon in induction of virus-augmented cytolysis. Increased lysis of YAC-HSV and PU51R-HSV was entirely due to interferon activation and was completely abolished by performing the 51Cr-release assay in the presence of anti-interferon serum. Because NC activity was not augmented by interferon, virus-enhanced NC lysis of M50-HSV and WEHI-HSV was not due to this nonspecific mechanism. Together, our data show that HSV-1 infection of NK/NC targets induces increased cytotoxicity, but the effector cell responsible for lysis is determined by the uninfected target, or by an interaction between the virus and target cell, rather than by a viral determinant alone.     

Purification and target cell range of in vivo elicited blast natural killer cells

Blast natural killer (NK) cells were elicited in the spleens of mice by treatments with the interferon inducers lymphocytic choriomeningitis virus (LCMV) or polyinosinic-polycytidylic acid (poly I:C). The blast-NK cells, separated on the basis of size by centrifugal elutriation, were compared with blast cytotoxic T lymphocytes (CTL) generated during infection with LCMV. In vivo treatments with antibody to asialo GM1 (AGM1) blocked the appearance of blast-NK cells but not blast-CTL. Antibody and complement depletion experiments indicated that the blast-NK cells were AGM1+, NK 1.2+/-, Lyt-5+/-, Thy+/-, Qa-5/NK 1.1+, Lyt-2-, B23.1-, and J11d-. Blast-NK cells could be unequivocally distinguished from blast-CTL, because the blast-CTL were completely sensitive to treatments with anti-Lyt-2 and complement, whereas the blast-NK cells were completely resistant. The blast-NK cells were purified from populations of large-size cells by antibody and complement treatments that depleted the co-eluting monocyte/macrophages and polymorphonuclear leukocytes. The population resulting after separation from dead cells over Percoll gradients represented approximately 1% of the total spleen cells, contained greater than 60% large granular lymphocytes and mediated greater than 15% killing of YAC-1 target cells in a 4-hr 51Cr release assay at an effector to target cell ratio of 1:1. The purified blast-NK cells lysed a broad range of target cells at relatively low effector to target cell ratios. The order of sensitivity of the target cells was YAC-1 much greater than K562 approximately equal to L-929 much greater than P815, consistent with that reported for NK cell-mediated lysis. The ability of the blast-NK cells to mediate lysis of NK cells also was examined. The purified NK cells mediated significant levels of lysis against the NK-like cloned line, NK1B6B10, in a 51Cr release assay. Furthermore, the purified blast-NK cells mediated lysis of bound blast-NK cells in a single-cell agarose assay. These results indicate that highly purified blast-NK cells are exceptionally efficient at mediating lysis and suggest that NK cells may act to negatively regulate the proliferation of NK cells by lysing other NK cells.     

Mechanism of defective NK cell activity in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex. II. Normal antibody-dependent cellular cytotoxicity (ADCC) mediated by effector cells defective in natural killer (NK) cytotoxicity

Our studies and other investigations have shown that NK effector cells can also mediate antibody-dependent cellular cytotoxicity (ADCC) through the use of the Fc gamma receptor on the NK cell membrane. Peripheral blood lymphocytes (PBL) derived from patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex exhibit a poor NK activity due to a defective "trigger" required for activation in the lethal hit stage of the NK lytic pathway. Consequently, it was important to delineate whether the defect in AIDS NK cells affected the ADCC function. By using the 51Cr-release assay, the ADCC cytotoxic activity of AIDS PBL was found to be within the normal range, despite the absence of significant NK activity. Several experiments corroborated that the same effector cells mediate both NK CMC and ADCC. Depletion of Fc gamma R-bearing cells resulted in elimination of both the ADCC and NK cytotoxic functions. Single cell analyses, using one- and two-target cell conjugates, revealed that the frequency of ADCC effector:target conjugates and the frequency of killer cells from AIDS PBL were comparable to the frequencies seen in the normal controls. However, when mixtures of NK and ADCC targets were used to form mixed two-target conjugates, the AIDS effector cells lysed only the bound ADCC target, whereas the normal effector cells lysed both the bound NK and ADCC targets. These results demonstrate clearly that the same NK/K effector cells from AIDS PBL, defective in NK activity, are not impaired in mediating ADCC activity. These findings were supported by the demonstration that AIDS PBL stimulated with ADCC targets, but not with NK targets, released NK cytotoxic factors, postulated mediators of the NK CMC reaction. These findings indicate that the NK/K cells in AIDS are triggered normally for ADCC activity but are not triggered for NK activity. Furthermore, the results indicate that the lytic machinery is not impaired in the AIDS NK/K cells.     

Preparation of target antigens specifically recognized by human natural killer cells

In an attempt to identify target cell membrane molecules recognized by natural killer (NK) cells, artificial membranes were prepared from detergent-solubilized plasma membranes of NK target cells and synthetic lipids. Such reconstituted membranes from human and rat NK target cells were shown to inhibit both human and rat NK-target cell conjugates in a species-specific fashion; these reconstituted membranes failed to inhibit NK cytotoxicity. The detergent-solubilized material from the human NK target K562 was subjected to various procedures prior to reconstitution and the conjugate inhibition assay. Conjugate inhibitory activity was lost upon trypsin digestion and incubation at 65 degrees C. This inhibition activity was absorbed to concanavalin A agarose and could subsequently be eluted with alpha-methylmannoside, resulting in approximately 20-fold purification. Gel filtration of this material on an AcA-34 column in detergent gave a broad activity peak with maximal activity in the molecular weight range of 30,000-165,000. Gel electrophoresis of purified membranes demonstrated multiple molecular weight bands in lipid membranes. The K562 membrane material, purified by concanavalin A agarose and gel filtration, inhibited conjugates between human NK cells and any of four human target cells, but not of conjugates with (1) human large granular lymphocytes and antibody-coated mouse tumor cells nor (2) rat NK cells and their target cells. Thus the purified glycoproteins from K562 retain the property of specific inhibition of human NK-target conjugates.     

Lipoxin A-induced inhibition of human natural killer cell cytotoxicity: studies on stereospecificity of inhibition and mode of action

Human leukocyte-derived lipoxin A (LXA; 5S,-6R,15S-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid) inhibits the cytotoxic activity of human natural killer (NK) cells. LXA and three of its isomers were prepared by total organic synthesis and assayed for activity with human NK cells. Dose-response studies showed that biologically derived LXA and synthetic LXA were equally effective in inhibiting NK cell cytotoxicity. 6S-LXA, with its 6S-OH group in an (S) configuration, proved to be approximately half as potent as LXA. In contrast, 6S-11-trans-LXA and 11-trans-LXA displayed virtually no inhibitory activities. The methyl esters of both LXA and 6S-LXA proved to be more potent than their corresponding free acids. Thus, LXA inhibition of NK cells displays clear-cut stereochemistry. In the absence of putative inhibitors, NK cells bind to their targets to form conjugates. This event is followed by polarization of the NK Golgi apparatus, which moves towards the plasma membrane that is in contact with the target cell. However, in the presence of either the methyl ester or free acid of LXA, the Golgi apparati of NK cells bound to their targets were randomly oriented. In contrast, neither 6S-11-trans-LXA nor the potent NK inhibitor prostaglandin E2 affected the polarization. Furthermore, although prostaglandin E2 resulted in a decrease in NK-target cell binding efficiency, LXA and its isomers failed to affect conjugate formation. Together these results indicate that LXA-induced inhibition of NK cytotoxicity does not act on NK cell binding but may block cytotoxicity by disrupting "signals" involved in the specific orientation of the Golgi. Thus, this latter event may appear to be important in cytotoxicity.     

Human killer cells and natural killer cells: distinct subpopulations of Fc receptor-bearing lymphocytes

Unstimulated human peripheral blood lymphocytes were depleted of K cells, which mediate antibody-dependent cellular cytotoxicity (ADCC) without removing NK cells, which mediate natural killing (NK). K cell depletion was achieved by buoyant centrifugation removal of lymphocytes that bound to glutaraldehyde-treated P815-AB cells at high lymphocyte-to-target ratios. Likewise, NK cells were removed with glutaraldehyde-treated K562 cells without removing K cells. Furthermore, both cytotoxic cell populations were observed directly in one agarose single-cell cytotoxic assay (ASCA) using P815-AB and K562 cells simultaneously as target cells. Moreover, the percentage of total cytotoxic cells was equal to the sum of the percentage of K and NK cells observed in separate ASCA. Collectively, these results indicate that K cells and NK cells are distinct subsets of FcR-bearing lymphocytes. One subset, K cells, has more avid Fc receptors (fcR) than NK cells and are 'activated' via thier FcR to kill antibody-coated target cells. The second subset, NK cells, have less avid FcR and are not 'activated' through their FcR to kill antibody-coated target cells.     

Dynamic redistribution of the activating 2B4/SAP complex at the cytotoxic NK cell immune synapse

The 2B4 molecule (CD244) has been described as a coreceptor in human NK cell activation. However, the behavior of 2B4 during the cytotoxic NK cell immune synapse (NK-IS) formation remains undetermined. In this study, we demonstrate the redistribution of 2B4 and the signaling adaptor molecule, signaling lymphocyte activation molecule-associated protein (SAP), to the cytotoxic NK-IS upon formation of conjugates between resting NK cells and EBV-infected 721.221 human cells. Confocal microscopy showed that 2B4 localized at the central supramolecular activation cluster, surrounded by a peripheral supramolecular activation cluster containing talin within NK cell and ICAM-1 on target cells. Videomicroscopy studies with 2B4-GFP-transfected NK cells revealed that 2B4 redistributed to cytotoxic NK-IS as soon as the cell contact occurred. Simultaneously, a SAP-GFP also clustered at the contact site, where it remained during the interaction period. The 2B4 molecular clusters remained bound to the target cell even after NK cell detachment. These results underscore the function of 2B4 as an adhesion molecule and suggest a relevant role in the initial binding, scanning of target cells, and formation of cytotoxic NK-IS. Finally, these findings are indicative of an important role of the activating 2B4/signaling lymphocyte activation molecule-associated protein complex during the recognition of EBV-infected cells.     

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.     

Natural killer cells can enhance the proliferative responses of B lymphocytes

In addition to lytic activity against malignant and virally transformed target cells, recent evidence has suggested that natural killer (NK) cells can modulate immune activities such as the suppression of B cell responses through noncytotoxic means. Using human B cells and highly purified autologous NK cells, we have demonstrated that NK cells can substantially augment the proliferative responses of B cells stimulated with the surface immunoglobulin crosslinking agents anti-IgM or Staphylococcus aureus Cowan strain I (SAC). This "enhancer" activity of NK cells was quite potent and was observed at an NK:B cell ratio as low as 0.05. Peak blastogenic responses of B cells cocultured with NK cells in the presence of B cell activators were observed at 2-3 days, similar to the responses of B cells in the absence of NK cells. Using the inhibitor of DNA synthesis mitomycin C, we determined that B cells and not NK cells were proliferating in cocultures of these lymphocytes stimulated with SAC. Activated B cells neither prevented the lysis of the isotope-labeled NK-sensitive target cell line K562 nor formed conjugates with NK cells, suggesting that cell contact was not a prerequisite for the effect. These studies have further expanded the functional repertoire of NK cells to include enhancer as well as suppressor and lytic activities.