Studies on the mechanism of natural killer cell-mediated cytotoxicity. VII. functional comparison of human natural killer cytotoxic factors with recombinant lymphotoxin and tumor necrosis factor

The present study was undertaken to evaluate the possible contribution of other cytokines to the lytic activity of NKCF-containing supernatants. We compared some of the functional properties of human NKCF and purified recombinant human rLT and rTNF. It was found that the target cell specificity of rLT was quite different from NKCF in that rLT was neither species specific nor NK specific. Furthermore, antibodies against rLT did not affect the lytic activity of NKCF. These results demonstrate that LT does not significantly contribute to the lytic activity mediated by NKCF. The target specificity of rTNF was found to be related to that of NKCF with the exception of one NK-resistant cell line that was lysed by rTNF in a 20-hr 51Cr-release assay. However, rTNF was not toxic to any of the target cells tested as assessed by trypan blue exclusion in a 20-hr assay unless the targets were labeled with 51Cr. In contrast, NKCF did kill target cells as detected by trypan blue exclusion that were not labeled with 51Cr. Further analysis of this mechanistic difference in the lytic activity of rTNF and NKCF revealed that rTNF in combination with either cycloheximide or mitomycin C but not IFN-gamma could lyse unlabeled U937 target cells. In addition, pretreatment of U937 target cells with nonradioactive Na2CrO4 at concentrations equivalent to that used to 51Cr-labeled cells resulted in their susceptibility to lysis by rTNF as assessed by trypan blue exclusion. These findings suggest that lysis of several susceptible target cells in 20 hr by rTNF requires the presence of additional agents that may be sublethally toxic and/or inhibitory to macromolecular synthesis. Antibody inhibition studies revealed that anti-TNF mediated from partial to complete inhibition of lysis of U937 by unfractionated supernatants containing NKCF. However, fractionation of such supernatants on chromatofocusing columns yielded two distinct peaks of activity eluting in the pH range of 5 to 6 and 7 to 8. Anti-TNF could inhibit the acidic form of NKCF but not the neutral form. It is concluded that NKCF activity is mediated in part by TNF or an antigenically related molecule as well as some other distinct factor(s). The lack of consistent inhibition of NK CMC by anti-TNF suggests that TNF alone is not sufficient to mediate NK activity, or else it is inaccessible to the added antibody.     

Studies on the mechanism of natural killer cell-mediated cytotoxicity. VI. Characterization of human, rat, and murine natural killer cytotoxic factors

Recent evidence has implicated natural killer cytotoxic factors (NKCF) as the lytic mediators of NK cell-mediated cytotoxicity reactions. The objective of this study was to examine and compare some of the biochemical and functional characteristics of human, rat, and murine NKCF. Supernatants containing NKCF were generated by stimulating effector cells with Con A or U937 (for human PBL) or YAC-1 (for rodent spleen cells) and tested for cytotoxic activity in a 20-hour (rodent) or 24-hour (human) 51Cr release assay. NKCF activity was inactivated by heating to 63 degrees C, 8 M urea, pH 2, and reduction and alkylation. These factors were highly sensitive to trypsin, moderately sensitive to papain and resistant to neuraminidase. Adsorption of human NKCF to U937 cells is inhibited by mannose-6-phosphate and adsorption of rodent NKCF to YAC-1 cells is inhibited by alpha-methyl-D-mannoside and fructose-6-phosphate. Oxidation of NKCF with sodium periodate abolished lytic activity. Pretreatment of NKCF with Con A but not pretreatment of target cells inhibited lytic activity. NKCF activity eluted in a single broad band of apparent MW of 15,000-40,000 after fractionation by HPLC gel permeating chromatography. Pooled fractions containing NKCF activity were subjected to some of the same tests performed on whole supernatants. Test result with semipurified NKCF confirmed that these factors are inactivated by trypsin or sodium periodate and that mannose-6-phosphate inhibits their binding to target cells. There were no major differences observed in NKCF produced by the three different species whether stimulated by Con A or NK-sensitive tumor cells. The evidence indicates that NKCF are glycoproteins in which disulfide bonding is essential for lytic activity. Furthermore, it appears that carbohydrate residues expressed on NKCF molecules are involved in the binding of these factors to the target cell membrane.     

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.     

Studies on the mechanism of specificity of human natural killer cells for tumor cells: correlation between target cell transferrin receptor expression and competitive activity

Previous studies to determine the nature of the specificity of natural killer (NK) cells for leukemic cells indicated that functional transferrin (Tf) receptors may be one of the determinants recognized by NK cells. To further investigate these observations, the relationship between cellular Tf receptor expression and ability to compete with a control K562 cell preparation in a standard chromium release assay was studied. K562 cells were selected at different phases of growth by removing cells from tissue culture at 1, 3, and 5 days postfeeding. Under these conditions, K562 cells, respectively, displayed relatively high, medium, and low numbers of Tf receptors and corresponding competitive activity against a control K562 cell preparation. K562 cells were modified by either trypsin, heat, or sodium butyrate (differentiation inducer) pretreatment. An NK-resistant clone was also studied. There was a good correlation between Tf receptor expression and cold competitive activity of the above K562 cell preparations (r = 0.82, P less than 0.01). The different tumor target cell lines, K562, Molt-4, Raji, HL-60, and MeWo, which would be expected to express different ranges of specificity, did not show a significant correlation between Tf receptor expression and their cold competitive activities against Cr-51-labeled K562 cells. Rabbit reticulocytes which express high numbers of Tf receptors were tested for their ability to compete with K562 cells for NK cells. These cells were able to compete with K562 cells while mature rabbit red blood cells which do not express Tf receptors did not compete well. These findings support the contention that the Tf receptor may be involved in NK cell recognition of some tumor cells.     

Studies on the mechanism of natural killer cell-mediated cytotoxicity. V. Lack of NK specificity at the level of induction of natural killer cytotoxic factors in cultures of human, murine, or rat effector cells stimulated with mycoplasma-free cell lines

We have proposed that lysis of target cells by NK cells is mediated by NK cytotoxic factors (NKCF). According to our model, for a target cell to be NK-sensitive, it must be recognized by the NK cell, it must stimulate the release of NKCF, and it must be sensitive to lysis by these factors. This report examines whether the ability to stimulate release of NKCF is a characteristic restricted to NK-sensitive tumor cells or whether it is also a property of NK-resistant target cells. Many different types of cell lines were tested for their ability to stimulate release of NKCF in the human, rat, and murine systems. It was found that mycoplasma-free NK-sensitive cell lines, resistant cell lines, and Con A could stimulate the release of NKCF. Many different types of cell lines grown in suspension or in monolayers were found to be effective stimulators, including T or B lymphoid, myeloid, and those of histiocytic origin. Cells cultured in the absence of serum stimulated NKCF release, thus ruling out the possible involvement of serum components in stimulation. NKCF was also produced by xenogeneic combinations of effector and stimulator cells, demonstrating lack of species specificity in NKCF production. Factors stimulated by NK-resistant cell lines or by Con A exhibited the same NK target specificity as supernatants stimulated by NK-sensitive tumor cells. The finding that many different NK-resistant cell lines can stimulate the release of NKCF indicates that there is no apparent NK specificity at the level of induction of NKCF release from human, rat, or murine effector cells. Therefore, the NK specificity of a target cell is determined ultimately by its sensitivity to lysis by NKCF.     

Human natural killer cytotoxic factor (NKCF): role of IFN-alpha

The relationship between production of NKCF and IFN-alpha by human lymphocytes was studied. NKCF activity was generated in response to K562-inducer cells. The presence of NKCF in supernatants was always accompanied by antiviral activity, but in several experiments IFN was detected without concomitant NKCF. In no instance was NKCF activity detected in the absence of IFN. Cell lines which were good inducers of IFN-alpha were found to be good inducers of NKCF. NKCF activity of supernatants was completely adsorbed after incubation with MOLT-4 cells, whereas there was only minimal depletion of IFN-alpha activity. Most of the antiviral activity and all of the NKCF activity of preformed supernatants was neutralized by anti-IFN-alpha serum, whereas anti-IFN-gamma serum and pH2 inactivation had minimal effect on either activity. Addition of IFN-alpha to neutralized supernatants restored NKCF activity. These experiments support the hypothesis that IFN-alpha is involved in the modulation of NKCF-lytic activity. Both antiviral and NKCF activities were abrogated when anti-IFN-alpha serum was added to cultures of lymphocytes plus inducer cells (induction phase). The addition of purified IFN-alpha to such cultures was effective in allowing resumption of NKCF activity; however, addition of IFN-gamma to these cultures did not overcome this block. The addition of purified IFN-alpha directly to supernatants generated in the presence of anti-IFN-alpha serum could not restore their NKCF activity, thereby suggesting an additional requirement for IFN-alpha in the production of NKCF. The possible role of IFN-alpha in the generation of NKCF and expression of its lytic activity is discussed.     

Comparison of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes

Precursors and effectors of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes are compared. Natural killer cells are resistant to gamma-irradiation (1000 R) whereas precursors of lymphokine-activated killer cells and cytotoxic T lymphocytes are sensitive. Lower doses of gamma-irradiation (500 R) remove precursors for cytotoxic T lymphocytes but not lymphokine-activated killer cells. In addition, lymphokine-activated killer cells are regenerated before classical CTL after sublethal doses of gamma-irradiation. Natural killer cells are resistant to anti-Thy 1 and C' and anti-thymocyte serum, but sensitive to anti-asialo GM1 and complement. Precursors of cytotoxic T lymphocytes are sensitive to anti-Thy 1 and complement and anti-thymocyte serum, but are resistant to anti-asialo GM1 and complement. Precursors of lymphokine-activated killer cells are partially sensitive to anti-Thy 1 and complement and anti-thymocyte serum, but are resistant to anti-asialo GM1 and complement. Effector cells of cytotoxic T lymphocytes are sensitive to anti-Thy 1 and complement and resistant to anti-asialo GM1 and complement. Lymphokine-activated killer cell effectors are sensitive to anti-asialo GM1 and complement at 24 hr after activation. These effectors are more closely aligned with classical natural killer effectors. Lymphokine-activated killer effectors, 7 days after activation, are resistant to anti-asialo GM1 and complement and sensitive to anti-Thy 1 and complement. Relationships and differences among these cytotoxic subsets are discussed.     

Studies on the mechanism of natural killer cytotoxicity. III. Activation of NK cells by interferon augments the lytic activity of released natural killer cytotoxic factors (NKCF)

The mechanism by which interferon (IFN) pretreatment of effector cells augments natural killer (NK) cell-mediated cytotoxicity (CMC) was examined by determining whether IFN has any effect on the production of natural killer cytotoxic factors (NKCF). NKCF are released into the supernatant of co-cultures of murine spleen cells and YAC-1 stimulator cells, and their lytic activity is measured against YAC-1 target cells. It was demonstrated that pretreatment of effector cells with murine fibroblast IFN or polyinosinic-polycytidylic acid (pIC) resulted in the release of NKCF with augmented lytic activity. Evidence indicated that the IFN-induced augmentation of NKCF activity required protein synthesis during the IFN pretreatment period, because concurrent pretreatment with both IFN and cycloheximide abrogated the IFN effect. Protein synthesis, however, is not required for the production of base levels of NKCF because emetine pretreatment of normal spleen cells did not result in a decrease in NKCF production. Furthermore, substantial levels of NKCF activity could be detected in freeze-thaw lysates of freshly isolated spleen cells. Cell populations enriched for NK effector cells, such as nylon wool-nonadherent nude mouse spleen cells, produced lysates with high levels of NKCF activity, whereas lysates of CBA thymocytes were devoid of NKCF activity. Pretreatment of spleen cells with either IFN or pIC resulted in an augmentation of the NKCF activity present in their cell lysates. Taken altogether, these findings suggest that freshly isolated NK cells contain preformed pools of NKCF. Pretreatment of these cells with IFN causes de novo synthesis of additional NKCF and/or activation of preexisting NKCF. According to our model for the mechanism of NK CMC, target cell lysis is ultimately the result of transfer of NKCF from the effector cell to the target cell. The evidence presented here suggests that the IFN-induced augmentation of NK activity could be accounted for by an increase in the synthesis, activation, and/or release of NKCF.     

Interleukin-1 alpha and tumor necrosis factor-alpha protect cells against natural killer cell-mediated cytotoxicity and natural killer cytotoxic factor

The protective effects of interferons (IFNs) against NK cell-mediated cytotoxicity (NK-CMC) is well established. We report here that both recombinant tumor necrosis factor-alpha (TNF-alpha) and recombinant interleukin-1 alpha (IL-1 alpha) can also protect some adherent target cells (e.g., the amniotic cells WISH and the cervical epithelial carcinoma cells HeLa-229) from NK-CMC in a dose-dependent manner. Like in the case of IFNs, the level of conjugate formation between target and effector cells (nonadherent peripheral blood lymphocytes) is not affected by pretreatment of the target cells with either TNF-alpha or IL-1 alpha. However, while the main effect of IFNs is to reduce the ability of target cells to stimulate the release of NK cytotoxic factor (NKCF) from effector cells, TNF-alpha and IL-1 alpha do not affect this process but rather reduce the target cell sensitivity to the lytic effect of NKCF. Therefore TNF-alpha and IL-1 alpha induce resistance to NK-CMC by a mechanism that differs from the one attributed to IFNs. The protective effect of TNF-alpha and IL-1 alpha is not mediated by the induction of IFN-beta 2/IL-6.     

Depressed natural cytotoxicity but normal natural killer cytotoxic factor (NKCF) production by mononuclear cells derived from patients with hepatocellular carcinoma

Summary This study investigated the relation between the production of natural killer cytotoxic factors (NKCF) and the phenomenon of natural killing (NK) activity against target K562 cells. Two different models of defective NK cell activity were employed. In the first instance, cytotoxic activity of mononuclear cells (MN) derived from patients with hepatocellular carcinoma was compared to the ability of these cells to produce NKCF. Although direct cytotoxicity was considerably impaired in these patients, the ability of their MN to produce NKCF when stimulated with K562 cells was found to be normal. In the second model, MN treated with the lysosomotropic drug monensin showed considerably reduced direct cytotoxic activity, although they were capable of producing normal amounts of NKCF when activated by K562 cells. These results therefore indicate that there is no correlation between NK activity and corresponding NKCF release, and suggest that NKCF production and activity is independent of direct NK cytotoxic activity.     

Decay-accelerating factor expression on either effector or target cells inhibits cytotoxicity by human natural killer cells.

Previous studies have shown that freshly isolated CD16+ NK cells are deficient in the expression of decay-accelerating factor (DAF), or CD55, a membrane regulator of C3 activation. In this study we investigated the significance, for NK cell-mediated lysis, of DAF expression on the target and effector cells. The effect of DAF expression on the susceptibility of NK cell targets was investigated by several means: first, DAF- cell lines were cloned from K562; second, the cloned DAF- cells were reconstituted with exogenous purified DAF; and third, anti-DAF F(ab')2 was used to block DAF function on the DAF+ K562 cells. Consistently, the presence of DAF in the target cell membrane, either naturally occurring or experimentally incorporated, afforded the target cell protection against lysis, and this protection could be blocked with anti-DAF. Similarly, targets for antibody-dependent cell-mediated cytotoxicity with exogenous DAF incorporated in their plasma membrane became less sensitive to antibody-dependent cell-mediated cytotoxicity by NK cells compared with the same target cells without incorporated DAF in their membranes. DAF incorporated in the plasma membranes of the effector NK cells made the NK cells less effective at killing K562 targets. The known function of DAF is to regulate C3 activation, and we were able to demonstrate that the isolated NK cell is capable of releasing C3. It is also possible that the participation of DAF in NK cell function represents a new, noncomplement-dependent function for DAF.     

Modification of the target cell surface with lipophilic glycoconjugates and interaction of the modified cells with natural killer cells

An experimental model system involving the modification of carbohydrate composition of the target cell surface with neoglycolipids was developed for studying the role of surface carbohydrates of target cells in the NK-cell-mediated cytotoxicity. The polymeric glycoconjugates of the Glyc-PAA-PEA and Glyc-PAA(Flu)-PEA types (where Glyc was an oligosaccharide residue, PAA poly(acrylamide) polymer, and PEA the phosphatidylethanolamine residue, and Flu fluorescein residue) capable of incorporation into the cell membrane were synthesized. The optimum structures of neoglycoconjugates and conditions for their incorporation into K562 and Raji cell lines, which differ in their sensitivity to the NK-cell-mediated lysis were selected. The mechanism of association of glycoconjugates with the plasma cell membrane and the kinetics of their elimination from the cell surface were investigated using the fluorescent-labeled Glyc-PAA(Flu)-PEA derivatives. The spatial accessibility of the carbohydrate ligands for the interaction with human NK cells was demonstrated. The target cells modified with the Le(x) trisaccharide were shown to be more sensitive to the cytotoxic effect of human NK cells than the intact cells. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.     

Association of decreased natural and antibody-dependent cellular cytotoxicity and production of natural killer cytotoxic factor and interferon in neonates

Cord blood lymphocytes (CBL) were compared with adult peripheral blood lymphocytes (a-PBL) for their: (i) natural killer (NK) and antibody-dependent cellular cytotoxic (ADCC) activities, (ii) target-binding capacity, (iii) ability to induce soluble natural killer cytotoxic factor (NKCF), (iv) interferon (IFN)-, interleukin 2 (IL-2)-, and lectin-induced augmentation of NK activity, and (v) ability to produce IFN against tumor targets in vitro. CBL depleted of adherent cells and Percoll-separated, NK-enriched subpopulations demonstrated significantly lower NK, ADCC, and target-binding activities compared to a-PBL. CBL produced significantly lower levels of NKCF directed against K562 tumor targets in comparison with a-PBL. Although the NK activity of CBL was not stimulated by either IFN or IL-2 to the same levels shown by a-PBL, the percentage enhancement of cytotoxicity of CBL by IFN and IL-2 was greater than that of a-PBL. Lectin-induced enhancement of cytotoxicity was significantly greater for CBL in comparison with a-PBL. Further, the ability of CBL lymphocytes to produce IFN-gamma in vitro against K562 target cells was significantly lower than that of adult PBL. These studies suggest an association between decreased NK, ADCC, and target-binding activities, induction of NKCF and IFN production by CBL, and increased susceptibility of neonates to infection.     

Suicide behavior of target cells after binding with natural killer cells

Human natural killer (NK) cell activity seems to be related to the integrity and function of the cytoskeletal apparatus. It has been hypothesized that microfilaments and microtubules play a pivotal role. In particular, the binding of the NK cell to the target cell requires microfilament integrity, and the lysis of bound targets seems to depend on microtubule assembly. We focused on the changes occurring in cytoskeletal elements and surface structures of NK cells and of target cells highly sensitive to NK activity (K562). Our observations, performed by fluorescence and scanning electron microscopy, besides confirming a rearrangement of the cytoskeletal apparatus in the effector cell, provide evidence that target cell cytoskeletal elements are involved in NK cell function. In K562 cells, after binding with NK cells, there is marginal rearrangement of actin and polarization of tubulin and vimentin in the contact regions, accompanied by modification of surface structures. These findings suggest that the target cell plays an active role in its own death by participating in the formation of an extended area of intimate contact with the killer cell. In addition, they lend credence to the surprising proposal that NK cells may induce a suicide mechanism in target cells.     

Detection of a perforin-like protein from the soluble cytotoxic factor of natural killer cells

The protein composition of the cytolytic factor (NKCF) secreted by natural killer cells was analyzed. Natural killer cells were isolated from spleens of nude rats and purified in the Percole gradient. SDS-PAAG analysis of NKCF revealed the presence of major proteins with an approximate molecular weight of 62-67 kDa. Biochemical properties of these proteins such as fractionation on a TSK-3000 SW gel filtration column, electrophoretical mobility in reduced conditions, enhanced cytolytical activity in the presence of Ca2+ demonstrate the similarity with the pore-forming protein--perforin. Apart from the major component, proteins with m. w. 42 kDa, 31-28 kDa, 17 kDa and 12-14 kDa were also found. It is possible that the Ca2+-independent cytolytical activity is bound to one of these minor protein components of NKCF.     

Differences in target cell DNA fragmentation induced by mouse cytotoxic T lymphocytes and natural killer cells

Fragmentation of YAC-1 target cell DNA during cytolysis mediated by mouse natural killer (NK) cells and cytotoxic T lymphocytes (CTL) was compared. Cleavage of nuclear chromatin was always an extensive and early event in CTL-mediated cytolysis, whereas with NK cell-mediated killing the degree of DNA fragmentation showed an unexpected relationship to the effector:target (E:T) ratio. At low NK:YAC-1 ratios, DNA fragmentation and 51Cr release were equivalent and increased proportionately until a ratio of about 50:1 was reached; at higher ratios, 51Cr release increased as expected but DNA fragmentation decreased dramatically. Comparison of time course data at E:T ratios producing similar rates of 51Cr release showed that the target cell DNA fragmentation observed in NK killing was not nearly as rapid nor as extensive as that observed with CTL effectors. These results suggest that NK cells induce target cell injury via two different mechanisms. One mechanism would involve lysis mediated by cell-to-cell contact, while the other may induce DNA fragmentation via a soluble mediator. In support of this notion, cell-free culture supernatants containing NK cytotoxic factor (NKCF) induced DNA fragmentation in YAC-1 cells. The DNA fragments induced by NK cells and NKCF-containing supernatants consisted of oligonucleosomes indistinguishable from those induced by CTL. The results presented here show distinct differences in target cell DNA fragmentation induced by CTL and NK cells, and suggest that these two effectors use different mechanisms to achieve the same end. CTL seem to induce DNA fragmentation in their targets by direct signaling, whereas NK cells may do so by means of a soluble factor.     

Radiosensitivity of human natural killer cells: binding and cytotoxic activities of natural killer cell subsets

The sensitivity of human natural killer (NK) cell activities (both binding and killing) after exposure of peripheral blood mononuclear cells to different doses of gamma radiation was studied. A panel of monoclonal antibodies was used to identify the NK and T-lymphocyte subsets and to evaluate their radiosensitivity. Peripheral blood mononuclear cells were irradiated with low (2-6 Gy) and high (10-30 Gy) doses and NK cell binding and cytotoxic activity against K562 target cells were studied after 3 h and 48 h in culture. The primary damage to NK cell activity was identified at the postbinding level and affected mainly the lytic machinery. After 48 h culture postirradiation, an overall depression of cytotoxic activity was observed, but ionizing radiation produced either a selection of the more cytotoxic NK cell subsets, which therefore might be considered more resistant to radiation damage than the less cytotoxic NK cells, or a long-term stimulation of cytotoxic activity in surviving cells.     

Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells.

Previously we have reported the purification and characterization of a novel cytokine from an EBV-transformed B cell line, RPMI 8866. This factor, termed natural killer cell stimulatory factor (NKSF), possessed pleiotropic activities including the induction of IFN-gamma from PBL, enhancement of cytotoxicity by NK cells, and stimulation of the proliferation of PBL. Purified NKSF was found to be a disulfide-linked heterodimeric protein composed of 35-kDa and 40-kDa subunits (p35 and p40). We now report the molecular cloning of cDNA for both subunits of NKSF from RPMI 8866 cellular RNA. The cDNA sequences indicate that both genes are novel, and Southern blot analysis confirmed that both cDNA are of human genomic origin. [35S]Methionine labeling indicated that cos-1 cells transfected with either p35 or p40 cDNA produced unique protein species of appropriate size. Methionine labeling of cos-1 cells cotransfected with p35 plus p40 cDNA yielded a broad band migrating between 70 and 90 kDa on a nonreducing gel. Reduction of this high molecular weight material yielded bands correlating with p35 and p40 gene products. Only culture supernatant from cotransfected cos-1 cells had a high level of NKSF biologic activity. That the high molecular weight material was responsible for this activity was indicated by the observation that biologic activity in the culture supernatant migrated at 70 to 90 kDa in a nonreducing gel. Furthermore, anti-p40 serum was able to block the biologic activities of both recombinant and natural NKSF, which indicates that it is a component of the active protein. In contrast, no activity could be detected in the supernatants of cos-1 cells transfected with p40 or p35 cDNA alone. The spectrum of biologic activity produced by cotransfected cos-1 cells was the same as NKSF purified to homogeneity from the RPMI 8866 cell line. A synergistic augmentation of some of these responses was found by the addition of IL-2 or the co-stimulators PHA or phorbol diester. The synergistic stimulation by NKSF plus IL-2 of T and NK function supports the possibility that these cytokines might prove useful in cancer therapy.     

Natural killer cell immunodeficiency in patients with chronic myelogenous leukemia. IV. Interleukin-1 deficiency, gamma-interferon deficiency and the restorative effects of short-term culture in the presence of interleukin-2 on natural killer cytotoxicity, natural killer-target binding and production of natural killer cytotoxic factor

We report herein that defective natural killer (NK) cell cytotoxicity, NK cytotoxic factor (NKCF) production and NK target binding ability of patients with chronic myelogenous leukemia (CML) are functionally restorable after short-term culture (less than 1 week) with recombinant interleukin-2 (rIL-2). We have previously reported that, despite normal to increased numbers of CD16+ large granular lymphocytes, fluorescence-activated-cell-sorted NK cells from CML patients are profoundly defective in NK cell activity and are unable to lyse the CML blast-crisis-derived, NK-sensitive target K562. Since we and others have also previously shown that the defective NK cytotoxicity from CML patients is restorable after 1-4 weeks of incubation with rIL-2, we therefore deemed it important to study the kinetics of IL-2-mediated NK restoration at earlier time intervals (less than 1 week). In the present report, we have demonstrated a significant restoration of NK cell cytotoxicity in CML patients against K562 after 5 days of short-term culture with rIL-2. In addition, recovery of NKCF production and restoration of target-binding capacity to normal levels by NK cells from CML patients were also observed after short-term (less than 1 week) rIL-2 treatment. Finally, we have demonstrated in the present report that adherent cells and peripheral-blood lymphoid cells from CML patients, as compared to normal controls, are unable to produce IL-1 beta and interferon-gamma, respectively, after stimulation with phorbol myristate acetate (IL-1 beta) and phytohemagglutinin-M (interferon-gamma).