The functional loss of human natural killer cell activity induced by K562 is reversible via an interleukin-2-dependent mechanism

In a recent study, we evaluated the functional status of human natural killer (NK) cells after their interaction with the NK-sensitive tumor target cell (TC), K562. We demonstrated that effector cells (EC), after treatment with K562 for 4 hr, lost greater than 90% of their original lytic activity. In this investigation, we examined whether this functional loss of NK cell activity represented an irreversible event in the NK lytic mechanism. Initial studies focused on the ability of K562-inactivated EC (ECi), which had been separated from their TC, to recover cytolytic activity following an 18-hr incubation. Our results indicated that ECi recovered 28% of their lytic activity in complete medium (CM) alone, 64% in CM containing interferon-beta (IFN-beta), and 91% in CM supplemented with interleukin 2 (IL-2). Analysis of the data revealed, however, that neither IFN-beta nor IL-2 simply boosted the lytic capacity of NK cells which initially escaped inactivation, but also, each cytokine affected the lytic capabilities of EC that were either truly inactivated by K562 or precursor NK (pre-NK) cells. Thus, to evaluate further the basis of IFN-beta and IL-2-induced ECi augmentation, we first treated the EC with IFN-beta or IL-2 prior to their interaction with K562 so that pre-NK cell subsets would be promoted to fully competent NK cells. Both pretreated EC preparations, after interacting with K562 for 4 hr, lost greater than 90% of their original lytic activities. NK inactivation did not result from cell death nor reflect alterations in conjugate formation or the percentages of Leu-7- and Leu-11-positive EC. IL-2-pretreated ECi, as did ECi, regained some lytic activity after incubation in CM alone, but recovered significantly more activity in CM containing IFN-beta or IL-2. In contrast to the restimulation profiles obtained for ECi and IL-2-pretreated ECi, IFN-pretreated ECi regained lytic function after incubation with IL-2, but not appreciably with IFN-beta or in CM alone. Overall, these findings suggest that EC, either untreated or pretreated with IFN-beta or IL-2, significantly lose their lytic capabilities following interaction with K562 while retaining their ability to bind to the TC; IFN-beta acts predominantly on pre-NK cells, but not on ECi; and IL-2 appears to play an important role in restoring lytic potential to functionally inactive NK cells.     

Target cell-directed inactivation and IL-2-dependent reactivation of LAK cells

In a previous study, we demonstrated that human natural killer cells (NK) lost their lytic activity after interaction with a sensitive target. The loss of NK activity also led to the loss of antibody-dependent cellular cytotoxicity (ADCC), prompting us to postulate that NK and ADCC activities may result from a common lytic mechanism. In this study, we examined whether nonadherent lymphocytes cultured 7 days in the presence of IL-2 (lymphokine-activated killer (LAK) cells) could also be inactivated and, subsequently, be reactivated in the presence of IL-2. We tested three populations of effector cells (EC): cells isolated from freshly drawn blood and tested immediately, cells cultured with IL-2 for 18 hr, and LAK cells. Once they have interacted with K562, all three cell populations lost greater than 90% of their NK-like lytic activity (NK-CMC) but only 80% of ADCC. However, when we treated the three cell types with antibody-coated K562, they lost 90-99% of NK-CMC and 90-97% of ADCC. In these inactivated effector cells we also observed: (i) a reduction in membrane expression of C-reactive protein; and (ii) a decrease in the expression of Leu-11a when EC were inactivated with antibody-coated K562. The loss of lytic activity against K562 was accompanied by a concomitant loss of activity against other LAK-sensitive targets as well as against antibody-coated targets (ADCC). In competitive inhibition experiments the inactivated effector cells failed to inhibit normal NK-CMC and ADCC activities mediated by fresh NK cells. As we have shown previously, this target-directed inactivation was not due to cell death or to lack of conjugate formation. Inactivated LAK cells regained their lytic potential when cultured with IL-2 and this effect was time dependent. By 72 hr, LAK cells inactivated with K562 regained 99% NK-CMC and 82% ADCC, whereas LAK cells inactivated with antibody-coated K562 regained only 80% NK-CMC and 70% ADCC. When we treated the effector cells with emetine, a potent inhibitor of protein synthesis, we could still inactivate the effector cells with K562 and with antibody-coated K562 but could not reactivate them with IL-2.     

Mechanism of action of phorbol myristate acetate on human natural killer cell activity

We have investigated the kinetics of inhibition and regeneration of human natural killer (NK) cell-mediated lysis of K562, a human erythroleukemia cell line, by the potent tumor-promoting agent phorbol-12-myristate-13-acetate (PMA). It is shown that PMA inhibits NK cell-mediated cytotoxicity (CMC) in a dose-dependent manner whether the compound is present throughout the 4-hr cytotoxic assay or the effector cells (EC) are pretreated with PMA. Pretreatment of the target cells (TC) with PMA produced a different profile of NK activity suggesting that PMA inhibition of NK-CMC is primarily due to the inactivation of EC. PMA-induced inhibition of NK-CMC does not affect TC binding and is not circumvented by compounds that enhance intracellular levels of cyclic guanosine monophosphate (cGMP) or calcium. Furthermore, and contrary to a recent report, PMA-induced inhibition of NK-CMC is independent of monocytes. Finally, kinetic studies revealed that PMA-induced inhibition of NK-CMC occurs rapidly and is fully reversible provided that “regenerated EC” are thoroughly washed, prior to the cytotoxic assay, to rid the cell suspension of residual PMA. The potential implications of these results to the currently accepted theory of TC destruction by NK cells, the stimulus-secretion model, are discussed.     

Phosphatidylinositol metabolism accompanies early activation events in tumor target cell-stimulated human natural killer cells

This study examined the role of phospholipid metabolism in human natural killer (NK) cells upon activation by tumor target cells(TC). The effector cell (EC) population consisted of peripheral blood lymphocytes enriched for NK cells. Upon a 5-min exposure of EC to the NK-sensitive tumor TC K562 and U937, nearly four- and threefold increases in the incorporation of 32P into phosphatidylinositol (PI) occurred, respectively. In contrast, no increase in 32P incorporation into PI was seen when two NK-resistant TC were used. In addition, little or no change in the incorporation of 32P into phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine took place with any of the above TC. Depletion of Leu 11b-positive cells abolished the increase in 32P incorporation into PI when K562 were used in the phospholipid assay. Furthermore, labeling kinetics of this phospholipid turnover showed that it occurred less than 5 min following exposure to NK-sensitive TC and that phosphatidic acid, a breakdown product of phosphoinositides, was produced during this 5-min period. These results indicated that metabolism of a phosphoinositide took place and that it occurred in association with early activation events in NK cells. Quercetin and dibutyryladenosine-cyclic monophosphate (dbcAMP) plus theophylline exerted profound inhibitory effects on both NK activity and PI metabolism, suggesting a linkage between the two events. The inhibitors had no effect on target cell-binding capacity, indicating that the inhibition occurred postbinding. PI metabolism took place in the absence of extracellular calcium even though NK activity was completely abolished under the same conditions. Thus, we have shown PI metabolism, but not other phospholipids, to occur in human NK cells upon exposure to NK-sensitive TC, in association with early activation events. This event was independent of extracellular calcium and could be inhibited by quercetin or dbcAMP plus theophylline.     

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

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

Inhibition of the calpain-mediated proteolysis of protein kinase C enhances lytic activity in human NK cells

Recent evidence from our laboratory has demonstrated that NK/LAK cell activation of human lymphocytes is protein kinase C (PKC)-dependent. Here, we have investigated the translocation of PKC in human NK cells exposed to sensitive targets or to PMA, a phorbol ester. In NK cells exposed to K562 for 6 hr, we observed a weak translocation of PKC whereas in NK cells exposed to PMA more than 90% of cytosolic PKC was translocated to the membrane in less than 5 min. Stimulation of NK cells with an NK-resistant target, however, did not translocate PKC even after 6 hr. Translocation of PKC to the membrane was followed by the appearance of PKM, the cytosolic calcium/phospholipid (Ca2+/PL)-independent form of PKC. The conversion of PKC to PKM was mediated by calpain, an intracellular calcium-dependent thiol proteinase. When we used two inhibitors of calpain, calpain inhibitor I (CI-I) and calpain inhibitor II (CI-II), both caused a dose-related enhancement of NK-CMC when the inhibitors were present throughout the 3-hr chromium release assay. This enhancement could be circumvented by PMA or by the PKC inhibitor H-7. CI-I and CI-II added together caused a greater increase in NK-CMC than when each was added alone. CI-I and CI-II also enhanced antibody-dependent cell-mediated cytotoxicity (ADCC), substantiating further our previous contention that the activation of both NK-CMC and ADCC may involve a common lytic pathway. Activation of NK cells with IL-2 for 18 hr at 37 degrees C was inhibited in the presence of CI-I. To investigate a possible feedback inhibition mechanism due to the buildup of PKC, we examined phosphatidylinositol (PI) metabolism in NK cells activated by IL-2 in either the presence or the absence of CI-I. We observed a significant decrease in PI turnover when NK cells, activated in the presence of IL-2 and CI-I, were stimulated with K562 as compared to NK cells activated by IL-2 alone, then stimulated with K562.     

Role of lipoxygenation in human natural killer cell activation

Nordihydroguaiaretic acid (NDGA), quercetin, eicosatetraynoic acid (ETYA), phenidone, and esculetin, agents known to inhibit cellular lipoxygenase (LO) activity, also inhibit human natural killer cell-mediated cytotoxicity (NK-CMC) of K562 tumor target cells (TC) in a dose-dependent fashion. Kinetic analysis demonstrated that LO inhibitors blocked an early event in the activation of the lytic mechanism but did not impair conjugate formation. LO inhibitors also did not affect subsequent chromium release, indicating that their site of inhibition was the NK cell and not the TC. The lipoxygenase products 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene-B4 significantly enhanced NK activity, with 5-HPETE being the more effective. Other LO products tested included 15-HPETE and the hydroxy derivatives 15-hydroxyeicosatetraenoic acid (15-HETE) and 5-HETE. These LO metabolites were either without effect on NK-CMC or inhibitory, depending upon the concentration. Additionally, we examined the ability of 5-HPETE to circumvent the effects of LO inhibitors and found that, in the presence of NDGA, ETYA or quercetin, 5-HPETE significantly (p less than 0.001) restored lytic activity. Inhibitors of LTB4 and LTC4 synthesis, diethylcarbamazine and U-60,257 respectively, produced no inhibition of NK activity. In fact, U-60,257 significantly (p less than 0.05) enhanced NK-CMC. Previous studies in our laboratory, with a new technique which allows for the separation of NK cells from K562 cells, have shown that K562-treated effector cells are greater than 90% inactivated when retested against fresh K562 in the standard chromium release assay. Lipids were extracted from K562-treated, Percoll-purified LGL and evaluated by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). No significant increases were seen in the arachidonic acid-derived LO products evaluated. Thus, our studies indicate that lipoxygenation may be required in the activation of NK-CMC, possibly as a means to generate oxygen radicals which have been previously implicated in NK-CMC.     

Inhibition of human natural killer cell activity by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine is an early but post-binding event

Recent evidence has demonstrated a protein kinase C (PKC)-dependent step in cytotoxic T lymphocyte activation. Here, we examined the influence of PKC in the lytic response of human NK cells to K562, an NK-sensitive tumor target cell. We used the known protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and HA1004. H-7 caused a dose-related inhibition of NK cell-mediated cytolysis (CMC) when the inhibitor was present throughout the course of the 3-h chromium release assay. The 50% inhibitory concentration for H-7 was 7 microM. In contrast, HA1004, which exerts a greater inhibitory effect on cyclic nucleotide-dependent protein kinases than PKC, had no effect on NK-CMC. The suppression of NK-CMC by H-7 was not due to inhibition of binding of the effector cells to target cells and could be reversed by the addition of PMA. H-7 was most effective in abrogating NK-CMC when added to the assay within the first 30 min and treatment of the effector and target cells with H-7 resulted in no loss of NK-CMC. Because nearly 50% of the normal NK lytic activity had taken place by 30 min, this suggested that H-7 inhibited an early event. H-7 exerted a dose-related suppression of antibody-dependent cell-mediated cytotoxicity (ADCC) suggesting that NK-CMC and ADCC share the utilization of PKC, however, HA1004 did not inhibit ADCC. Treating NK cells with IL-2 or IFN-beta did not overcome the inhibition of NK-CMC by H-7. In this study, we have thus demonstrated the presence of a PKC-dependent step in NK-CMC and ADCC.     

Studies on the mechanism of natural killer cytotoxicity. II. coculture of human PBL with NK-sensitive or resistant cell lines stimulates release of natural killer cytotoxic factors (NKCF) selectively cytotoxic to NK-sensitive target cells

This investigation has employed the "innocent bystander" type of experimental design to determine whether soluble cytotoxic factor(s) are released during interactions between human peripheral blood lymphocytes (PBL) and NK-sensitive target cells. PBL cocultured with NK-sensitive Molt-4 or K562 target cells in the lower well of a miniaturized Marbrook culture released natural killer cytotoxic factors (NKCF), which diffused across a 0.2-mu Nucleopore membrane and lysed Molt-4 or K562 target cells cultured in the upper chamber. Coculture of PBL with the NK-resistant Raji or WI-L2 cell lines also induced release of NKCF. These factors were selectively cytotoxic to NK-sensitive targets and lysed Molt-4 and, to a lesser extent, K562 cells. However, Raji, WI-L2, and RPMI 1788 cells were all resistant to lysis. In addition, low density fractions from Percoll density gradients that were enriched for NK effector cells also released increased levels of NKCF during coculture with Molt-4 cells. Lysis of Molt-4 and K562 targets was observed after exposure to NKCF for 48 hr and 60 to 70 hr, respectively. Cellfree supernatants containing NKCF were obtained after a short time of incubation (i.e., within 5 hr of coculture of PBL with NK target cells). The factors were nondialyzable, stable at 56 degrees C for 3 hr, and showed partial loss of activity on storage at 4 degrees C or -20 degrees C for 7 days. These data suggest that NKCF may be involved in the lytic mechanism of human NK cell-mediated cytotoxicity.     

Effect of Fas+ and Fas− Target Cells on the Ability of NK Cells to Repeatedly Fragment DNA and Trigger Lysis via the Fas Lytic Pathway

We and others have recently shown that human NK cells express the Fas ligand (FasL) constitutively and that they can trigger the lysis of Fas positive (Fas+) target cells (TC) by apoptosis. We have also previously demonstrated that NK cells exposed to sensitive TC temporarily lose their ability to lyse sensitive TC via the granule-mediated pathway and that this loss is recovered when inactivated NK cells (NKi) are incubated in medium supplemented with IL-2, IL-12 or IL-15. In this study, we investigated the fate of the Fas-lytic pathway in NK cells exposed to either Fas+ or Fas– TC. To this end, we exposed NK cells to Jurkat (Fas–) or Jurkat (Fas+) TC for up to 6 h, separated NK cells from the TC and assessed the residual lytic activity against K562, a traditional human NK cell target, Jurkat Fas+ and Jurkat Fas– TC. Fas lytic activity was determined in calcium free medium, in the presence or absence of two distinct Fas-blocking monoclonal antibodies and a Fas.Fc fusion protein. In parallel experiments, the extent of DNA fragmentation in the three TCs was also assayed by the JAM test. Our results indicate that: (i) NK cells exposed to susceptible Fas+ TC temporarily lose most of their lytic potential due to the granule-mediated pathway, while only partially losing the Fas-lytic pathway. They also partially lose their ability to fragment DNA. (ii) NK cells exposed to Fas+ TC completely recover the Fas lytic pathway and the ability to fragment DNA via the Fas/Fas ligand when incubated in medium supplemented with IL-2 for 18 h.     

Antigenic, functional, and molecular genetic studies of human natural killer cells and cytotoxic T lymphocytes not restricted by the major histocompatibility complex

Cytotoxicity not restricted by the major histocompatibility complex (MHC) is mediated by two distinct types of lymphocyte: natural killer (NK) cells and non-MHC-restricted cytotoxic T lymphocytes (CTL). These two types of cytotoxic lymphocytes can be distinguished by antigenic phenotype, function, and molecular genetic studies. In human peripheral blood, NK cells are identified by expression of the Leu-19 and/or CD16 cell surface antigens, and lack of CD3/T cell antigen receptor (Ti) complex expression (i.e., CD3-,Leu-19+). Peripheral blood non-MHC-restricted CTL express both CD3 and Leu-19 (i.e., CD3+, Leu-19+, referred to as Leu-19+ T cells). Both Leu-19+ T cells and NK cells lyse "NK-sensitive" hematopoietic tumor cell targets, such as K562, without deliberate immunization of the host. However, most "NK activity" in peripheral blood is mediated by NK cells, because they are usually more abundant and more efficient cytotoxic effectors than Leu-19+ T cells. The cytolytic activity of both NK cells and Leu-19+ T cells against hematopoietic targets was enhanced by recombinant interleukin 2 (rIL 2). NK cells, but not peripheral blood Leu-19+ T cells, were also capable of lysing solid tumor cell targets after short-term culture in rIL 2. Southern blot analysis of NK cells revealed that both the T cell antigen receptor beta-chain genes and the T cell-associated gamma genes were not rearranged, but were in germ-line configuration. These findings indicate that NK cells are distinct in lineage from T lymphocytes and do not use the T cell antigen receptor genes for target recognition.(ABSTRACT TRUNCATED AT 250 WORDS)     

The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes

We examined the antigenic and functional characteristics of human peripheral blood lymphocytes that differentially express the CD16 (Leu-11) and Leu-19 (NKH-1) antigens. Leu-19 is a approximately 220,000 daltons protein expressed on approximately 15% of freshly isolated peripheral blood lymphocytes. Within the Leu-19+ subset, three distinct populations were identified: CD3-,CD16+,Leu-19+ cells; CD3+,CD16-,Leu-19+ cells; and CD3-,CD16-,Leu-19bright+ cells. Both the CD3+,CD16-,Leu-19+ and CD3-,CD16+,Leu-19+ populations mediated non-major histocompatibility complex (MHC)-restricted cytotoxicity against the NK-sensitive tumor cell K562 and were large granular lymphocytes. CD3-,CD16+,Leu-19+ NK cells were the most abundant (comprising approximately 10% of peripheral blood lymphocytes) and the most efficient cytotoxic effectors. The finding that CD3+,Leu 19+ lymphocytes mediated cytotoxicity against K562 unequivocally demonstrates that a unique subset of non-MHC-restricted cytotoxic CD3+ T lymphocytes are present in the peripheral blood of unprimed, normal individuals. However, CD3+,CD16-,Leu-19+ cells comprised less than 5% of peripheral blood lymphocytes, and the cytotoxic activity of this subset was significantly less than CD3-,CD16+,Leu-19+ NK cells. Most CD3+,Leu-19+ T cells co-expressed the CD2, CD8, and CD5 differentiation antigens. The antigenic and functional phenotype of peripheral blood CD3+,Leu-19+ cytotoxic T lymphocytes corresponds to the interleukin 2-dependent CD3+ cell lines that mediate non-MHC-restricted cytotoxicity against NK-sensitive tumor cell targets. A small population of Leu-19bright+ lymphocytes lacking both CD3 and CD16 was also observed. This population (comprising less than 2% of peripheral blood lymphocytes) contained both large agranular lymphocytes and large granular lymphocytes. CD3-,CD16-,Leu-19bright+ lymphocytes also mediate non-MHC-restricted cytotoxicity. The relationship of these CD3-CD16-,Leu-19bright+ lymphocytes to CD3+ T cells or CD16+ NK cells is unknown.     

Natural killer cell inhibition of young spherules and endospores of Coccidioides immitis

The recent development of a method for culturing the parasitic form of Coccidioides immitis by using conditions compatible with the growth of lymphoid cells has enabled us to investigate the role of natural killer (NK) cells in defense against this pathogenic fungus. Pure cultures of spherules and endospores were grown in RPMI 1640 which contained 10% calf serum. Single cell suspensions of young spherules and endospores were incubated in the presence of freshly isolated human peripheral blood lymphocytes (PBL). After a 4-hr incubation, the colony-forming ability of the fungus was significantly reduced. Leu-11 is a monoclonal antibody that binds to the Fc receptor of NK cells. When PBL were incubated in the presence of this monoclonal antibody and complement, the colony-forming ability of C. immitis was not reduced, indicating that the effector cell involved in reduction of colony-forming units is also recognized by the Leu-11 monoclonal antibody. Classical NK activity can be enhanced by preincubation with interferon; the inhibitory activity of the PBL which are responsible for the reduction in colony-forming units of C. immitis is similarly enhanced by pretreatment with interferon. When PBL are incubated in the presence of young spherules and endospores for 24 hr, the cellfree supernatants will kill U937 target cells. In addition to stimulating the release of NK cytotoxic factor, C. immitis is susceptible to inactivation when incubated in the presence of factors released by PBL which have been incubated in the presence of either K562 or C. immitis. Other evidence reported by this laboratory demonstrates that C-reactive protein is present on the surface of NK cells and that antibody to this molecule blocks NK-mediated killing of standard tumor cell targets. Pretreatment with anti-C-reactive protein also blocks the ability of PBL to inhibit the colony-forming capacity of this fungus. These data suggest that the cell that inhibits the in vitro growth of the pathogenic fungus, C. immitis, is an NK cell.     

Lysis of human solid tumor cells by lymphokine-activated natural killer cells

The ability of NK cells to lyse noncultured solid tumor cells was investigated, and the results were compared with lysis of K562. Purified NK cell fractions separated by either Percoll centrifugation or a cell sorter exhibited higher level of lysis against noncultured melanoma cells than did NK-depleted cell fractions. However, the level of lysis was low (less than 10% lysis). Adding recombinant interleukin 2 (rIL 2) to the 4-hr assay induced significant lysis (more than 10%) of noncultured melanoma cells in 18 of 23 (78%) Percoll-enriched NK cell fractions and seven of 11 (64%) sorted Leu-11a+ cells at an E:T ratio of 80 and 10, respectively. In contrast, only two of 13 (14%) PBMC, five of 17 (29%) Percoll-decreased NK cell fractions, and one of 12 (8%) sorted Leu-11a- cells lysed noncultured melanomas in the presence of rIL 2. rIL 2 induced NK cells to lyse noncultured lung and breast cancer cells, as well as melanoma tumors. Exposure of NK cells to 2000 rad radiation abrogated the rIL 2-induced cytotoxicity against noncultured melanomas. Preculture of PBMC for 18 hr with recombinant interferon-gamma (rIFN-gamma) resulted in a modest level of lysis of non-cultured melanomas by sorted Leu-11a+ cells. Adding rIL 2 to the assay increased the cytotoxic activity in both rIFN-gamma-activated Leu-11a+ and Leu-7+ NK subsets. The level of noncultured tumor lysis correlated well with that of K562 lysis in all of the experiments. Purified NK cell fractions in rIL 2 cultures increased cytotoxic activity against noncultured tumor cells with incubation time for up to 3 days, and the level of NK cell-mediated lysis was dependent on both doses of rIL 2 and length of incubation. In contrast, both NK-depleted and sorted Leu-11a- cells demonstrated very low levels of solid tumor lysis after 3-day cultures with a high dose of rIL 2. Killer cell precursors induced by 3-day cultures of sorted cell fractions with rIL 2 and rIFN-gamma were found in both Leu-11a+ and Leu-7+ NK subsets, but not Leu-4+ or Leu-3a+ T lymphocytes. These results indicate that NK cells become cytotoxic for noncultured solid tumor cells by a brief contact with rIL 2, and increase cytotoxic activity after culture with rIL 2.     

Identification of a human natural killer cell target cell antigen with monoclonal antibodies

mAb have been derived against NK cell-sensitive target cells in an effort to identify the target cell structure involved in Ag recognition by NK cells. Several mAb were selected for further study based on their preliminary target cell binding characteristics. Flow cytometry demonstrated that each of these mAb bound to a series of NK-sensitive target cells of various origins (e.g., K562 and Molt-4) while having little or no reactivity with several NK-resistant target cell lines (e.g., SB and Daudi). Functional studies revealed that two of the mAb were able to inhibit the lysis of NK-sensitive K562 target cells by freshly isolated, endogenous NK cells in a dose-dependent fashion. Further, these mAb also could inhibit the killing of K562 target cells by both activated NK cells and cultured lymphokine-activated killer cells, as well as the cytolysis of other NK-sensitive target cells by each of these effector cell populations. Control experiments with another mAb which bound to the target cells but did not inhibit lysis implied that the effects of these mAb on NK cell function was not the result of steric hindrance. Single cell conjugate assays demonstrated that the mAb inhibited NK cell lysis via the inhibition of binding (recognition). Biochemical analysis of this target cell structure revealed that it was a molecule of approximately 42 kDa which may exist as a homodimer in its native state. Thus, it appears that the mAbs identify an unique Ag on the surface of NK cell-sensitive target cells which is involved in NK cell Ag recognition.     

Natural killer (NK)-like cytotoxic activity of allospecific T cell receptor-gamma,delta+ T cell clones. Distinct receptor-ligand interactions mediate NK-like and allospecific cytotoxicity

We recently generated a series of human alloantigen-specific, CD3+,TCR-gamma,delta+ clones by stimulating CD3+,CD4-,CD8- T cells from normal individuals with allogeneic lymphoblastoid cell lines (LCL). As reported previously, these clones display cytotoxic activity against their specific stimulators but not against irrelevant LCL. Further studies of these and other TCR-gamma,delta+ clones, described in this report, indicate that most but not all of these clones express the NK cell associated marker, NKH-1 or Leu-19, and kill NK-sensitive targets such as the K562 and Molt 4 lines, but not an irrelevant LCL or NK-resistant line, Raji. TCR-gamma,delta+ clones which lacked expression of Leu-19 lysed their allospecific targets but had no detectable NK activity. The allospecific cytotoxicity of Leu-19+ and Leu-19- clones was inhibited by mAb to CD3 or the TCR delta-chain. In contrast, the NK-like activity of the Leu-19+ clones was enhanced by these antibodies over a wide range of antibody concentration. Although mAb to LFA-1 markedly inhibited both the allospecific and NK-like activity of these clones, an HLA class I framework specific mAb (W6/32) had no effect on NK-like cytolysis but did inhibit allospecific killing, suggesting that the target structures on the surface of allospecific and NK-sensitive cells are distinct. The receptors utilized by the TCR-gamma,delta+ clones to recognize NK-sensitive and allospecific targets are also distinct, since killing of NK-sensitive targets was blocked by the presence of cold (unlabeled) NK-sensitive cells but not by cold allospecific targets, whereas allospecific cytolysis was inhibited by cold allospecific targets but not by NK-sensitive cells. We conclude that some CD3+,TCR-gamma,delta+ clones exhibit NK-like as well as allospecific killing and that these two activities are mediated by distinct receptor-ligand interactions.     

Monoclonal antibody against K562 cell line accelerates killing of the target cells by large granular lymphocytes

A monoclonal antibody (MoAb 11-4) was raised against K562, a human erythroleukemia cell line sensitive to natural killer cell-mediated cytotoxicity (NK-CMC). Immunological analysis revealed MoAb to be IgG_2b. Alone, the MoAb was not cytotoxic for K562 and did not bind to the effector cells, but the addition of this antibody to macrophage-depleted human peripheral blood lymphocytes increased killing of K562 in a 4-hr NK-CMC assay. The maximum increase in NK-CMC was observed when MoAb 11-4 was added to target cells prior to the formation of effector/target cell conjugates. This effect was dose dependent, was specific for K562, and, contrary to conventional antisera, occurred at very low concentrations of MoAb. When MoAb was added either to Percoll-purified large granular lymphocytes (LGL) or to LGL-depleted lymphocytes, only the latter demonstrated a significant increase in the killing of K562 in a 4-hr chromium release assay. Kinetics studies revealed that although the overall LGL-mediated lysis was only slightly increased at 4 hr, the maximum lytic activity was reached within 2 hr. These studies suggest that (1) human LGL and LGL-depleted cell populations bear Fc receptors for mouse IgG_2b and (2) although the cytotoxic activities of both cell populations are increased by treatment with MoAb 11-4, the kinetics of this increase are different.     

Mechanism of cell contact-mediated inhibition of natural killer activity

Natural killer cell activity is inhibited by primary cultures of monolayer cells. In this study, we analyzed the mechanism of the inhibition. Inhibited NK cells showed unaltered binding capacity to NK sensitive K562 cells. The orientation of the effector cells' actin-containing microfilaments, an event known to occur during the programming for the lysis stage in lytic conjugates, was unaffected by the inhibition. In single cell cytotoxicity experiments, the number of killer cells among conjugate-forming cells was reduced. The capacity of the inactivated NK cells to secrete cytotoxic factors upon stimulation with Con A was also impaired. Both NK-resistant inactivating target cells and NK-sensitive K562 cells were sensitive to the toxic factors secreted by NK cells. Thus, the results indicate that the target cell-mediated inactivation of NK cell is based on a block in the lethal hit stage, possibly due to reduced release of toxic factor(s) from the effector cells. The capacity of inactivated effector cells to mediate antibody-dependent cellular cytotoxicity was unimpaired, suggesting that the contact-mediated inhibition of cytotoxicity selectively affects NK cells.     

Detection of double target binders at the single cell level: an evaluation of the specificity of NK and MLC-induced NK-like cells

MLC-generated cells were tested on 7 consecutive days in the single cell cytotoxicity assay to determine the kinetics of natural and allospecific killing. Maximum cytotoxicity to the NK-sensitive target, K562, was found on Day 3 of MLC with an increase at that time in both the number of cells binding and the number of cells killing K562. The maximum allospecific response was found on Days 6 and 7 with an increase in cells able to bind and kill the alloantigen-bearing target. To determine whether the anti-K562 and allospecific killing were mediated by the same effector cells or different cell populations, both targets were tested simultaneously in the single cell assay. At no time during the 7 days were cells detected capable of simultaneously binding both K562 and allospecific targets. These data indicate that there are two different cell populations responsible for allospecific cytotoxicity and MLC-induced NK-like cytotoxicity. The cytotoxic specificity of unstimulated and MLC-generated NK-like cells was also investigated. When two different NK-sensitive targets (e.g., K562 and MOLT-4) were tested together in the single cell assay, there was no concurrent binding of targets by either fresh PBL prior to MLC stimulation or Day 3 MLC-generated cells. When unstimulated effector cells were enriched for NK activity by Percoll density gradient centrifugation, only a small number of effector cells simultaneously binding two different NK-sensitive targets was detected in the single cell assay. These results imply that the NK cell population is heterogeneous and composed of subpopulations recognizing diverse target specificities.     

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.