Induction of human lymphokine-activated killer cells by IFN-alpha and IFN-gamma

By traditional definitions, NK cells can be activated by cytokines to exhibit two functionally distinct levels of cytotoxicity. Whereas IL-2-mediated activation of NK cells leads to the development of lymphokine-activated killer (LAK) cytotoxicity, characterized by the acquisition of cytolytic activity against NK-resistant targets, IFN-treated NK cells become activated without the acquisition of novel cytolytic specificities. In this study we show that NK cells activated by 18 to 24 h of stimulation with either IFN-alpha or IFN-gamma do acquire LAK cytolytic activity, demonstrated by the ability of IFN-treated PBMC to lyse NK-resistant COLO 205 cells as well as fresh tumor targets. The level of IFN-alpha-induced LAK activity was significantly greater than that induced by IFN-gamma, although IL-2-induced LAK activity was considerably greater than IFN-alpha-induced LAK cytotoxicity. Maximal IFN-induced LAK cytotoxicity occurred after 24 h of culture, and occurred with the use of IFN-alpha at 500 U/ml and IFN-gamma at 1000 U/ml. Whereas neutralizing antibody experiments demonstrated that IFN-alpha-induced LAK activation did not involve the participation of endogenously produced IL-2, the partial inhibition (63%) of IFN-gamma-induced LAK cytotoxicity by anti-IL-2 and of IL-2-induced LAK by anti-IFN-gamma (33.3%) indicates that the induction of LAK cytotoxicity by either of these individual cytokines involves the endogenous production and participation of the other cytokine. Similar to IL-2-induced LAK cells, phenotypic analysis revealed that IFN-alpha/gamma LAK cells were Leu-19+, although the Leu 19"dim"+ subset exhibited greater IFN-induced LAK activity than the Leu-19"bright"+ subset. The results of this study clearly demonstrate that IFN-alpha and IFN-gamma induce classic LAK activity and IFN-gamma plays a participatory role in the optimal induction of LAK cells by IL-2.     

Inhibition of murine lymphokine-activated killer (LAK) cell activity by adherent cells

The effects of adherent cell depletion, indomethacin, and prostaglandin E2 (PGE2) on murine LAK cell activity were investigated. Removal of plastic adherent cells from splenocyte suspensions either prior to 5-day culture with 1000 U/ml of recombinant human IL-2 (rIL-2) or prior to assay resulted in an enhanced LAK cell cytotoxicity compared to that of whole spleen cell suspensions. Indomethacin enhanced LAK cell cytotoxicity of whole splenocyte suspensions if present during the culture period, but had no effect on whole splenocyte or adherent cell-depleted cell suspensions if added just prior to assay. PGE2 suppressed LAK cell activity of nonadherent splenocyte but not whole splenocyte suspensions when present during the culture period. In vivo treatment of mice with indomethacin enhanced cytotoxicity directed toward both LAK sensitive, natural killer (NK) resistant (P-815) and LAK, NK sensitive (YAC-1) tumor cell targets. Splenocytes from indomethacin-treated mice cultured with additional indomethacin and rIL-2 exhibited highest LAK cell activity. The results from this study indicate that LAK cells are regulated by adherent cells which suppress LAK cell activity. This suppression can be reversed both in vitro and in vivo by indomethacin. This study has important implications for the possible clinical use of indomethacin in the potentiation of in vivo and in vitro LAK cell activity for immunotherapeutic protocols.     

Natural killer and lymphokine-activated killer cell activities from human marrow precursors. II. The effects of IL-3 and IL-4

Both IL-3 and IL-4 have multi-CSF activity on early marrow progenitors. We have examined the effect of IL-3 and IL-4 on the differentiation of NK cells from their marrow-derived precursors and have further examined the interactions of these cytokines with IL-2 and IL-1. We tested marrow which had been depleted of mature cells and of E rosette-positive cells (including NK cells) by treatment with soybean lectin and SRBC (SBA-E-BM). The cytolytic activities of the SBA-E-BM samples were tested in 51Cr-release assays after 7 days of liquid culture. K562 targets were used as a measure of NK activity and NK-resistant Daudi targets were used to measure lymphokine-activated killer (LAK) cell activity. Neither NK nor LAK activity was detectable in marrow cultured in medium without cytokines, or in medium containing IL-3, or IL-4 alone. Both of these cytokines were shown to be inhibitory to the IL-2-induced generation of NK and LAK activity from SBA-E-BM at concentrations as low as 1 U/ml. The inhibitory activity of both IL-3 and IL-4 was found to occur early in the marrow cultures, with little or no inhibitory effects seen if added 48 h after IL-2. IL-3 appeared to be specifically inhibitory to NK cell precursors since addition of IL-3 to cultures of PBMC did not inhibit IL-2-induced lytic activities. In contrast, IL-4 was equally inhibitory to the activation of marrow and peripheral blood NK cells by IL-2. Mixing experiments demonstrated that the reduced lytic activity in IL-3 or IL-4 containing marrow cultures were not due to suppression of the NK effectors, nor could marrow cultured in IL-3 or IL-4 serve as targets for IL-2-activated NK cells. Phenotype analysis of the lymphoid cells in marrow cultures containing IL-2 combined with IL-3 or IL-4 revealed fewer cells expressing Leu-11 (CD16), or Leu-19 (CD56) and fewer CD16, CD56 coexpressing cells compared with marrow cultured in medium containing IL-2 alone. The inhibitory activity of IL-4, but not IL-3, could be partially reversed if IL-1 was added to the cultures, suggesting that IL-1 and IL-4 have opposing activities on NK cells responsiveness to IL-2. These interactions between cytokines might be important in the regulation of NK cell differentiation and on the functional activity of mature NK cells.     

Human lymphokine-activated killer (LAK) cells: III. Effect ofl-phenylalanine methyl ester on LAK cell activation from human peripheral blood mononuclear cells: Possible protease involvement of monocytes,natural killer cells and LAK cells

Summary We have shown that depletion of monocytes from human peripheral blood mononuclear cells (PBMC) byl-phenylalanine methyl ester (PheOMe) enhanced lymphokine-activated killer cell (LAK) generation by recombinant interleukin-2 (rIL-2) at high cell density. In this study, we have investigated the mechanism of action of PheOMe on LAK activation by using trypsin, chymotrypsin, tosylphenylalaninechloromethanol (TPCK, a chymotrypsin inhibitor), tosyl-l-lysinechloromethane (TLCK, a trypsin inhibitor), phenylalaninol (PheOH), and benzamidine. PBMC were treated with 1–5 mM PheOMe for 40 min at room temperature in combination with the various agents, washed and assessed for their effects on natural killer (NK) activity against K562 cells and monocyte depletion. The treated cells were then cultured with or without rIL-2 for 3 days. LAK cytotoxicity was assayed against⁵¹Cr-labeled K562 and Raji tumor target cells. TPCK at 10 µg/ml partially inhibited depletion of monocytes by PheOMe. TLCK did not prevent depletion of monocytes nor inhibition of NK activity induced by PheOMe. TPCK and TLCK inhibited NK activity by themselves. TPCK but not TLCK inhibited rIL-2 induction of LAK cells. On the other hand, PheOH and benzamidine (analogs of PheOMe) lacked any effect on monocyte depletion but abrogated the inhibitory effect of PheOMe on NK activity. They had no effect on rIL-2 activation of LAK activity enhanced by PheOMe. Trypsin potentiated the inhibitory effect of PheOMe on NK activity and monocyte depletion. Trypsin partially inhibited IL-2 activation of LAK activity enhanced by PheOMe. Chymotrypsin had little effect on NK activity but prevented the inhibitory effect of PheOMe on NK activity. It had little effect on monocyte depletion induced by PheOMe. PheOMe was hydrolysed by monocytes and chymotrypsin to Phe and methanol as determined by HPLC. TPCK inhibited hydrolysis of PheOMe by monocytes. Our data suggest that the effects of PheOMe on monocytes, NK cells and LAK activation involve protease activities of monocytes.     

Lymphokine-activated killer cells in rats: generation of natural killer cells and lymphokine-activated killer cells from bone marrow progenitor cells

The coculture of rat bone marrow cells with recombinant interleukin-2 induced the generation of cells mediating natural killer (NK) activity and subsequent lymphokine-activated killer (LAK) activity depending upon the dose of IL-2 and time of culture. NK activity was detected as early as 4 to 5 days after the addition of IL-2 and could be evoked with as little as 5 to 50 U/ml. The induced NK cells had large granular lymphocyte (LGL) morphology and expressed 0X8 and asialo GM1 surface markers but did not express 0X19 or W3/25 markers. LAK activity was detected only after 5 days of culture, and required above 100 U/ml IL-2. Cells mediating LAK activity also expressed 0X8 and asialo GM1 but not 0X19. The generation of detectable NK and subsequent LAK activity was due to induction of early progenitor cells and not contaminating mature LGL/NK cells within the bone marrow population since of removal of such mature NK cells with L-leucine methyl ester (L-LME) did not affect the subsequent generation of either activity. Moreover, the removal of actively dividing cells as well as mature NK cells from the bone marrow by treatment with 5-fluorouracil (5-FU) in vivo enriched the remaining bone marrow population for both NK and LAK progenitor cells. The phenotype of the L-LME- and 5-FU-resistant NK and LAK progenitor cells within populations of bone marrow was determined by antibody plus complement depletion analysis. Although treatment of normal bone marrow with anti-asialo GM1 + C reduced the induction of NK and LAK activity in 5-day cultures, treatment of 5-FU marrow with anti-asialo GM1 + C did not affect either activity. Treatment with a pan-T cell antibody + C did not affect the development of NK or LAK activity under any conditions. Thus, the 5-FU-resistant NK/LAK progenitors were asialo GM1 negative but became asialo GM1+ after induction by IL-2. Finally, evidence that bone marrow-derived LAK cells were generated directly from the IL-2-induced NK cells was obtained by treating the IL-2-induced LGL/NK cells with L-LME.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lymphokine-activated killer cells in rats. IV. Developmental relationships among large agranular lymphocytes, large granular lymphocytes, and lymphokine-activated killer cells

The developmental relationships among large agranular lymphocytes (LAL) large granular lymphocytes (LGL) and the activation of these cells into lymphokine-activated killer (LAK) cells by rIL-2 was investigated. Highly enriched populations of LAL were isolated from Fischer 344 spleen cells by a combination of nylon-wool filtration (to remove B cells and macrophages), treatment with a pan T cell antibody plus complement (to remove T cells) and incubation in L-leucine methyl ester (to remove LGL). The resultant cells were highly enriched in morphologically identifiable LAL which expressed asialo GM1 and partially expressed the OX8 surface marker. The enriched LAL did not contain detectable NK cytotoxic activity, did not express pan T cell (OX19), Ia, Ig, or laminin surface markers and contained less than 0.2% LGL. Incubation of LAL in a low dose of rIL-2 (100 U/ml) induced the generation of LGL having NK activity within 24 h of culture. Longer culture periods (48 h) resulted in a continued increase in the percentage of LGL and higher levels of NK activity. However, with this low dose of rIL-2, little or no LAK activity (i.e., reactivity against NK-resistant target cells) was generated. With a high dose of rIL-2 (500 U/ml), LAL responded by first generating LGL with NK activity (within 24 h), with subsequent generation of LAK activity by 48 h. Evidence that the development of granular lymphocytes from LAL was responsible first for NK activity and then LAK activity was demonstrated by depletion of the generated granular NK or LAK effector cells by second treatments with L-leucine methyl ester. Concomitant with the induction of LGL with NK or LAK activity, rIL-2 also caused LGL to proliferate and expand four- to five-fold in 48 h. This occurred in the presence of high or low dose rIL-2. These results indicate that LAL are the precursors of LGL/NK cells, that LAL, LGL/NK cells and LAK cells appear to represent sequential developmental or activation stages and that LAL may comprise major source of LAK progenitors in lymphoid populations having few LGL or mature active NK cells.     

Inhibition of in vitro LAK generation by OK-432

Summary The present study was designed to investigate the in vitro effect of OK-432 on interleukin-2-(IL-2) induced lymphokine activated killer (LAK) generation, and especially to test whether OK-432 can substitute for IL-2 or act in synergism with IL-2 for activation of cytotoxic lymphocytes. Surprisingly, our results showed that the addition of OK-432 to 4-day LAK activation cultures significantly inhibited both the generation of cytotoxic effectors to the natural killer (NK) resistant Daudi cell line and the proliferative responses of lymphocytes in a dose dependent manner. The inhibition of activation was total at 0.5 KE/ml of OK-432, a dose which was still effective in augmenting NK activity against K562. The addition of penicillin G potassium (PCGk), which is contained in OK-432 at a concentration of 134,700 units/mg of dried cocci, to the LAK culture system also inhibited LAK generation at equivalent concentrations as contained in the OK-432 preparation. This inhibition of LAK generation by OK-432 was significantly eliminated by dialysis of OK-432. These results indicated that the inhibition of LAK generation was partly due to PCGk contained in the OK-432 preparation, and that OK-432 did not act synergistically with IL-2 in standard LAK activation systems.     

Prostaglandin E2-mediated suppression of murine lymphokine-activated killer cell activity generated from tumor-bearing hosts by interferon-gamma

The effect of mouse recombinant interferon-gamma (IFN-gamma) on murine lymphokine-activated killer (LAK) cell activity was investigated using a natural killer-resistant, LAK-sensitive, spontaneously developed, weakly immunogenic, syngeneic murine mammary adenocarcinoma, a tumor model mimicking that of human disease. When all of the splenocytes prepared from tumor-bearing mice were cultured with recombinant interleukin-2 (IL-2) and IFN-gamma, LAK cell activity was suppressed in an IFN-gamma dose-dependent manner. An increase in the prostaglandin E2 (PGE2) content in the corresponding culture media was detected, as was IFN-gamma dose dependent. The suppression of generation of LAK cell activity by IFN-gamma was abrogated, accompanied by the elimination of the increase in PGE2 content, when plastic dish and nylon wool-treated nonadherent macrophage-depleted splenocytes were used. These results indicated that IL-2-induced LAK cell activity generated from the splenocytes of tumor-bearing mice was suppressed by IFN-gamma, and that PGE2 secreted from the macrophages of the splenocyte cultures served as the mediator in this IFN-gamma dose-dependent suppression of IL-2-induced LAK cell activity.     

Generation of lymphokine-activated killer cell activity from non-NK precursor cells

It is possible to generate high levels of lymphokine-activated killer (LAK) activity in short-term culture from cells enriched for natural killer (NK) activity. To determine whether LAK activity can also be generated from non-NK cells, we have depleted peripheral blood lymphocytes (PBL) of NK cells prior to culture with IL-2. NK activity in PBL is correlated with the intensity of staining with the lysosomotropic vital dye quinacrine. Quinacrine dim PBL, which are devoid of lytic NK cells, are capable of developing LAK activity following culture with IL-2. We have also separated PBL using the NK-associated NKH-1 marker. Depleting NKH-1+ cells eliminates NK activity but the ability to develop LAK activity is retained. NKH-1-depleted cells generate less LAK activity than unseparated or NKH-1-positive cells and do not proliferate as well as unseparated cells to IL-2. When NK-depleted cells are subsequently examined for the expression of the NKH-1 antigen, this marker is absent from most cells at Day 3 of IL-1 culture, but is expressed on an increasing number of cells by Days 6-8. These results suggest that LAK derived from non-NK cells is functionally and phenotypically similar to LAK from PBL-containing NK cells, and may be the result of the activation of an NK precursor population.     

TNF-alpha and IFN-gamma reverse IL-4 inhibition of lymphokine-activated killer cell function

Recombinant IL-4 inhibits IL-2-induced lymphokine-activated killer (LAK) cell development of PBMC. We evaluated the effect of various cytokines in reversing IL-4-mediated LAK inhibition. PBMC were cultured in IL-2 (10-1000 u/ml) with or without IL-4 (2-100 u/ml) and tested for cytotoxicity against the NK-sensitive K562 cells and NK-resistant UCLA-SO-M14 cells. Addition of IL-4 at the beginning of culture suppresses LAK activity in a dose-dependent fashion. Addition of IFN-gamma or TNF-alpha partially reverses IL-4-mediated inhibition (30-100%) in a dose-dependent fashion. IFN-gamma and TNF-alpha must be added within the first 24 hr of initiating culture in order to reverse IL-4 inhibition. Furthermore, IFN-gamma and TNF-alpha are most effective at reversing IL-4 inhibition at low concentrations of IL-2 (less than 100 u/ml). Addition of other IL-2-induced cytokines such as GM-CSF (50 u/ml), M-CSF (250 u/ml), and IFN-alpha (10-10,000 u/ml) fails to reverse IL-4 inhibition. In addition to suppression of LAK induction, IL-4 also inhibits IL-2-induced IFN-gamma and TNF-alpha protein production in PBMC. The reversal of IL-4-mediated LAK inhibition by TNF-alpha and IFN-gamma may therefore be due to resupply of these endogenously suppressed cytokines.     

Targeted deletion of CD44v7 exon leads to decreased endothelial cell injury but not tumor cell killing mediated by interleukin-2-activated cytolytic lymphocytes

In the current study, we investigated the nature and role of CD44 variant isoforms involved in endothelial cell (EC) injury and tumor cell cytotoxicity mediated by IL-2-activated killer (LAK) cells. Treatment of CD44 wild-type lymphocytes with IL-2 led to increased gene expression of CD44 v6 and v7 variant isoforms and to significant induction of vascular leak syndrome (VLS). CD44v6-v7 knockout (KO) and CD44v7 KO mice showed markedly reduced levels of IL-2-induced VLS. The decreased VLS in CD44v6-v7 KO and CD44v7 KO mice did not result from differential activation and expansion of CD8+ T cells, NK, and NK-T cells or from altered degree of perivascular lymphocytic infiltration in the lungs. LAK cells from CD44v7 KO mice showed a significant decrease in their ability to adhere to and mediate lysis of EC but not lysis of P815 tumor cells in vitro. CD44v7-mediated lysis of EC by LAK cells was dependent on the activity of phosphatidylinositol 3-kinase and tyrosine kinases. Interestingly, IL-2-activated LAK cells expressing CD44hi but not CD44lo were responsible for EC lysis. Furthermore, lysis of EC targets could be blocked by addition of soluble or enzymatic cleavage of CD44v6-v7-binding glycosaminoglycans. Finally, anti-CD44v7 mAbs caused a significant reduction in the adherence to and killing of EC and led to suppression of IL-2-induced VLS. Together, this study suggests that the expression of CD44v7 on LAK cells plays a specific role in EC injury and that it may be possible to reduce EC injury but not tumor cell killing by specifically targeting CD44v7.     

Differential sensitivity of cytotoxic T lymphocytes and lymphokine-activated killer cells to inhibition by L-ornithine

The selective inhibition of murine cytotoxic T lymphocyte (CTL) differentiation in C57B1/6 (B6) anti-DBA/2 mixed leukocyte cultures (MLC) by the amino acid L-ornithine (Orn) could not be reversed by addition of up to 1000 U/ml IL-2. Analysis of the effects of Orn on induction of lymphokine-activated killer (LAK cells), using dosages of IL-2 from 10-1000 U/ml and measuring cytolytic activity against two tumor targets (P815 and YAC-1) over the course of 5 days, indicated that LAK cells were not suppressed by Orn. LAK precursors and effector cells were CD8- and ASGM1+, indicating that they were derived from natural killer (NK) cells. We also found that the growth and maintenance of cloned CTL lines were not sensitive to inhibition by Orn; nor was their acquisition of nonspecific cytolytic activity in the presence of high lymphokine concentrations. Thus, induction of naive CTL shows differential susceptibility to Orn inhibition relative to LAK and LAK-like activities by NK and cloned CTL lines in response to IL-2.     

The human lymphokine-activated killer cell system. V. Purified recombinant interleukin 2 activates cytotoxic lymphocytes which lyse both natural killer-resistant autologous and allogeneic tumors and trinitrophenyl-modified autologous peripheral blood lymphocytes

Culture of human peripheral blood lymphocytes (PBL) in purified natural or recombinant interleukin 2 in the absence of exogenous antigen or mitogen causes the differentiation of nonlytic precursor cells into lymphokine-activated killers (LAK). A titration of purified Jurkat IL-2 (BRMP, FCRC, NIH) IL-2 showed that the relatively low concentration of 5 U/ml was optimal for LAK activation. When the responding PBL were pretreated with either mitomycin C or gamma irradiation, LAK activation did not occur, indicating that proliferation, in addition to differentiation, is required. The spectrum of target cells susceptible to LAK lysis in a 4-hr chromium-51-release assay includes fresh NK-resistant tumor cells and trinitrophenyl (TNP)-modified autologous PBL. Unmodified PBL are not lysed. Cold target inhibition studies indicated that LAK lysis of autologous TNP-PBL is totally inhibited by fresh tumors cells, and that tumor lysis is inhibited by TNP-PBL. Additionally, allogeneic tumors totally inhibit lysis of autologous tumor cells in other cold target studies. These results demonstrate that the lytic activity expressed by LAK is not HLA restricted, is not limited to tumor cells, and is "polyspecific" as indicated by the cross-reactive recognition of multiple target cell types in these cold target inhibition studies.     

In vivo administration of low-dose human interleukin-2 induces lymphokine-activated killer cells for enhanced cytolysis in vitro

We have examined the effect of the intradermal administration of IL-2 on the generation of natural killer (NK) cell and lymphokine-activated killer (LAK) cell activity. Peripheral blood mononuclear cells (PBMC) obtained from borderline lepromatous (BL) and lepromatous leprosy (LL) patients and normal volunteers prior to and after IL-2 injection were stimulated in vitro with IL-2 and their cytolytic activities compared against 51Cr labeled target K562 cells, Daudi cells, and monocytes. Before IL-2 administration, PBMC obtained from BL/LL patients and normal volunteers possessed similar levels of NK cell activity indicating that the NK cell activity of the BL/LL patients was intact. LAK cell activity was induced with IL-2 in vitro in both BL/LL patients and in normal volunteers. The level of LAK cell activity in BL/LL patients was, however, suboptimal. A single intradermal dose of 25 micrograms IL-2 had no effect on the phenotype of circulating mononuclear cells in either patients or normal volunteers. However, 6-12 days after IL-2 injection and subsequent restimulation of the PBMC with IL-2 in vitro, cytolytic activity of LAK cells obtained from the BL/LL patients was enhanced while cells from normal volunteers expressed the same high levels of activity as observed before IL-2 injection.     

Murine trophoblast can be killed by lymphokine-activated killer cells

The ability of fetal trophoblast cells in the placenta to resist cell-mediated lysis may be important for successful pregnancy. Previous studies in this laboratory demonstrated that cultured midterm mouse trophoblast cells are not susceptible to allospecific CTL generated by standard in vitro protocols, to antibody-dependent cell-mediated cytotoxicity, or to naive or IFN-activated NK cells, despite expressing the requisite target structures. However, we now report that murine trophoblast can be killed, in a non-MHC-specific manner, by LAK cells. Normal mouse spleen cells cultured for 4 days in IL-2-containing lymphokine preparations characteristically killed both NK-sensitive (YAC-1) and NK-resistant (EL4, P815) target cells, and mediated significant lysis of both cultured and freshly isolated trophoblast cells (35 to 55%, E/T 100/1). Pretreatment of the LAK cells with anti-ASGM1 antibody and C markedly reduced the lysis of trophoblast and YAC-1 targets, suggesting that the responsible cells belonged to the NK lineage. The ability of IL-2-activated NK cells to kill midterm murine trophoblast cells was confirmed using a population of highly lytic NK cells generated by culturing spleen cells from severe combined immunodeficiency mice in 500 U/ml rIL-2 for 5 days. These effector cells killed YAC-1, EL4 and P815 target cells at much lower E/T ratios than was achieved with the normal splenic LAK cells, and mediated significant lysis of both freshly isolated (45 to 50%, E/T 20/1) and cultured trophoblast cells (68 to 76%, E/T 20/1). The susceptibility of trophoblast to LAK cells and IL-2-activated NK cells supports the need for suppressor mechanisms regulating IL-2 activity at the maternal-fetal interface.     

Interferon is able to reduce tumor cell susceptibility to human lymphokine-activated killer (LAK) cells

It is known that IL-2 induces lymphocytes to produce interferon-gamma (IFN-gamma) and this IFN type is particularly efficient in inducing tumor cell resistance to natural killer (NK) cell-mediated lysis. We have investigated the effect of IFN on tumor cell sensitivity to LAK cell-mediated cytotoxicity. Pretreatment of the human K562 leukemia and HHMS melanoma with IFN-gamma and the Daudi lymphoma with IFN-alpha caused a significant reduction in sensitivity to lysis by human LAK cells generated in vitro in the presence of human recombinant IL-2 (100 U/ml). The LAK activity was mediated by cells expressing NK cell markers (CD16,NKH1) as well as by cells with T cell markers (CD3, CD5). IFN-treated K562 cells were protected from lysis mediated by all these populations. Supernatants from LAK cultures containing IFN-gamma were able to induce NK and LAK resistance when used to pretreat K562 overnight. Antibodies to IFN-gamma but not to IFN-alpha were able to neutralize this activity. Taken together, these results indicate that the production of IFN-gamma by LAK cells may be of importance in induction of tumor cell resistance to LAK cell-mediated lysis.     

Modulation of perforin, granzyme A, and granzyme B in murine natural killer (NK), IL2 stimulated NK, and lymphokine-activated killer cells by alcohol consumption.

Alcohol consumption in mice suppresses the cytolytic activity of natural killer (NK) and lymphokine-activated killer (LAK) cells through unknown mechanisms. Herein, we found that alcohol consumption decreased target cell-induced release of granzyme A activity in freshly isolated splenic NK cells, in NK cells stimulated for 18 h with 1000 IU/ml of interleukin 2, and in LAK cells. The total activity and protein expression of granzymes A and B also were lower in these cells than in cells isolated from water-drinking mice. Interleukin 2 increased granzyme A protein expression independent of alcohol consumption; however, this increase was associated with decreased enzyme activity. In contrast, granzyme B protein expression and enzymatic activity increased in response to interleukin 2. Perforin activity and protein expression were reduced in LAK cells generated from alcohol-consuming mice. We conclude that the mechanism underlying the suppression of NK and LAK cytolytic activity by alcohol consumption involves the collective reduction of target-induced release, activity, and expression of perforin and granular proteases.     

Regulation of NK cell activity by prostaglandin E2: the role of T cells

The influence of T cells on the production of prostaglandins (PGE2) and on PGE2-mediated regulation of natural killer (NK) activity was studied. Supernatants from peripheral blood mononuclear cells (PBMC) and from PBMC depleted of T cells ((PBMC)-T), both of which had been incubated in plastic petri dishes overnight, contained similar amounts of PGE2, as detected by radioimmunoassay and by their potential to inhibit NK activity of peripheral blood mononuclear cells in a 51Cr release assay with K 562 cells as the target population. However, the NK activity of PBMC was inhibited significantly more strongly (P less than 0.005) by PGE2-containing supernatants than was the NK activity of (PBMC)-T. In further assays, in which synthetic PGE2 in concentrations of 10(-4) and 10(-5)M was added, a significant inhibition of NK activity was observed in PBMC populations (P less than 0.05), but not in (PBMC)-T. Thus, T cells did not seem to be involved in the control of PGE2 production, but their presence was necessary for PGE2-mediated inhibition of NK activity.     

Potentiated lymphokine-activated killer cell activity generated by low-dose interleukin-2 and mismatched double-stranded RNA

Summary Lymphokine-activated killer (LAK) cell activity was measured in human peripheral blood mononuclear cells (PBMC) treated in vitro for 3 days with recombinant interleukin-2 (rIL-2) and mismatched double-stranded RNA (dsRNA). Lytic activity was measured utilizing K562 (NK-sensitive) and 786-0 (NK-resistant) target cells. PBMC cultured with rIL-2 (10–1000 BRMP U/ml) alone showed concentration-dependent lytic activity against the 786-0 target cells, while cells cultured in unsupplemented medium or medium supplemented with mismatched dsRNA (200 µg/ml) alone could not lyse the 786-0 targets. The combination of mismatched dsRNA with suboptimal concentrations of rIL-2 (10–30 U/ml) showed enhancement of both natural killer (NK) and LAK cell activities. The uptake of [³H]thymidine by treated effector cells was dependent on time and rIL-2 concentration and was not increased in the cells treated with low-dose rIL-2/mismatched dsRNA, compared to those treated with low-dose rIL-2 or mismatched dsRNA alone. Similarly, changes in the expression of CD3, CD4, CD8, CD57, CD16 and CD25 cell surface antigens were dependent on rIL-2 concentration and not altered by the presence of mismatched dsRNA. These results indicate that mismatched dsRNA can potentiate rIL-2-induced LAK cell activity by increasing the functional activity per cell, rather than by increasing the number of activated cells.