Anti-tumor cytotoxic potential and effect on human bone marrow GM-CFU of human LAK cells generated in response to various cytokines.

The effects of various recombinant cytokines i.e. IL-1 alpha, IL-3, IL-4, IL-6, IFN-gamma, TNF-alpha and GM-CSF used either alone or in combination with IL-2, were investigated in this study. First, their capacity to induce killer cells from human PBL was examined by evaluating the degree of killing of human NK-sensitive K562 or NK-resistant Daudi cells. Second the effects of these cytokines, LAK cells (at 1/1, 2/1, 4/1 ratio LAK effectors/bone marrow cell targets) and of the supernatants from washed killer cell cultures, were examined on the colony forming ability of human bone marrow for GM-CFU in vitro. Various degrees of NK activity against K562 was observed in PBL stimulated with the cytokines, whereas LAK activity was found only with IL-2 alone. Culture of PBL with IL-2 + IL-1 alpha or IL-2 + IL-6 or IL-2 + GM-CSF resulted in the highest LAK killing. However, addition of TNF-alpha, or IFN-gamma to IL-2 in cultures resulted in a significant suppression of LAK cell activity. Addition of IL-1 alpha, IL-2, IL-3, and IL-4 to BM cultures had little or no effect on day 14 GM-CFU, whereas addition of IL-6 and GM-CSF resulted in a stimulatory effect. LAK cells induced with IL-2 alone had no significant suppressive effects on GM-CFU.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preadministration of a T-suppressor factor enhances tumor immunity in DBA/2 mice

Summary Human peripheral blood mononuclear cells (PBM) activated with recombinant interleukin-2 (IL-2) generate potent lytic activity (LAK) against a variety of malignant cells. IL-2 alone is sufficient for LAK generation, but high concentrations are needed to generate optimal cytotoxicity. Our recent studies based on combinations of biological agents indicated that alternative activation pathways may exist. Synergy for LAK induction was investigated using IL-2 and tumor necrosis factor- (TNF). Single-cell suspensions of primary human lung carcinomas were prepared from seven established cell lines and 32 fresh tumor specimens. Not only were all cell lines sensitive to allogeneic LAK, but also all fresh tumors were sensitive to some degree to both autologous and allogeneic LAK lysis measured by a 4-h ⁵¹Cr-release assay. LAK-mediated cytotoxicity, induced with a combination of human recombinant IL-2 (Cetus, 100 U/ml) and TNF (Genentech, 500 U/ml), showed a mean fourfold increase (range 0.7–16.3) over IL-2 alone. No lytic activity was generated from PBM incubated with media or TNF alone. The sequence dependence of adding IL-2 and TNF in enhancing cytolytic activity was also studied. In vitro kinetics data revealed that the addition of TNF 2–6 h before the addition of IL-2 greatly increased LAK activity over that obtained from the simultaneous addition of the two cytokines. These results demonstrated (a) the synergy of IL-2 and TNF for generating LAK; (b) the lysis of fresh primary lung cancer cells by LAK; and (c) the sequence dependence of IL-2 and TNF for the induction of optimal LAK activity.This work was supported by NCI Grants RO2-CA45225 and CAO 9611-01, and by an award from the Prouss Foundation     

Susceptibility of autologous target cells to lysis by lymphokine-activated effectors from interferon-α-treated chronic myelogenous leukaemia patients

Summary Chronic myelogenous leukemia (CML) patients in chronic phase display compromised lymphokine-activated killer (LAK) cell induction, which is partly restored after therapy with interferon . However, the relative resistance of the leukemic cells from these patients to autologous or allogeneic LAK lysis is not affected by this treatment. In an attempt to render CML cells more susceptible to lysis or cytostasis, they were precultured in serum-free medium with or without recombinant growth factors. In eight patients studied, interleukin-3 (IL-3) significantly enhanced the spontaneous short-term (6-day) proliferation of CML cells, with retention of ability to form colonies in methylcellulose. Culture in either medium alone or IL-3 led to a significant enrichment of CD14⁺ and CD33⁺ cells but to a reduction in CD34⁺ cells. In contrast, culture of the same cells in IL-2 (to generate autologous LAK activity) resulted in a loss of CD14⁺ and CD33⁺ as well as CD34⁺ cells but in a significant increase in CD3⁺ and CD56⁺ cells. Despite similarities in their phenotypes, IL-3 cultured cells but not those cultured in medium alone acquired susceptibility to lysis by the IL-2-cultured autologous LAK cells. These results may have significance for the design of novel combination immunotherapy in CML.This work was supported in part by the Deutsche Forschungsgemeinschaft (SFB 120)     

IL-4 regulates IL-2 induction of lymphokine-activated killer activity from human lymphocytes

IL-4 is a pluripotent lymphokine acting on various cell types. We investigated the role of human IL-4 on the generation of lymphokine-activated killer (LAK) activity. Human IL-4 alone did not induce LAK activity and inhibited IL-2 induction of LAK activity from unstimulated PBMC, peripheral blood null cells, spleen cells, and lymph node cells in a dose-dependent manner. IL-4 also inhibited several phenomena induced by IL-2 such as cell proliferation, augmentation of NK activity, increase of Leu-19+ cells, and expression of IL-2R(p55) on either CD3+ or Leu-19+ cells. IL-4, however, augmented cell proliferation with other T cell mitogens including PHA, Con A, PMA, or allo-MHC Ag with or without IL-2. In contrast to unstimulated cells, IL-4 alone induced marked cell proliferation and LAK activity as well as Leu-19+ cells from in vitro IL-2 preactivated PBMC or null cells, and did not inhibit IL-2 induced cell proliferation, LAK activity, Leu-19+ cells and IL-2R(p55) expression, but rather augmented them with low doses of IL-2. Although IL-4 alone induced LAK activity from peripheral blood of some patients previously given IL-2, IL-4 inhibited in vitro LAK generation with IL-2 from these cells in most cases. Therefore, IL-4 appears to directly inhibit the IL-2 activation pathway via IL-2R(p70) and prevent resting LAK precursors from proliferating and differentiating into final effector cells. However, once cells were sufficiently preactivated by IL-2, IL-4 induced LAK activity and did not inhibit IL-2 activation of these cells. These data suggest an immunoregulatory role of IL-4 on human null cells and T cells.     

Interleukin-6 enhances the induction of human lymphokine-activated killer cells

Summary Human peripheral blood mononuclear cells develop a powerful lytic capacity when cultured in vitro with interleukin-2 (IL-2), becoming lymphokine-activated killer cells (LAK cells). As part of an investigation into means of influencing this process, the effect of other cytokines has been examined. In this study we describe the ability of interleukin-6 (IL-6) to regulate the induction and function of human LAK cells. The results show that substitution of IL-6 for IL-2 did not lead to the development of functional LAK cells, nor was IL-6 able to alter the lytic capacity of established LAK cells. However, when IL-6 was included with IL-2 during the induction phase of the LAK cells, the resulting cells displayed considerably greater lytic activity than those prepared with IL-2 alone. This effect was IL-6 dose-related. These results indicate that LAK cell development may be positively regulated in vitro; the implications of this observation for the clinical usage of LAK cells are discussed.     

Immunomodulation in patients receiving intravenous Bryostatin 1 in a phase I clinical study: comparison with effects of Bryostatin 1 on lymphocyte function in vitro

Summary Bryostatin 1 is a protein kinase C activator that inhibits growth of tumour cells and activates lymphocytes in vitro, properties that have encouraged its use in phase 1 clinical studies as an anticancer agent. We investigated interleukin-2(IL-2)-induced proliferation and lymphokine-activated killer (LAK) cell activity in peripheral blood mononuclear cells (PBMC) from cancer patients receiving Bryostatin intravenously. After Bryostatin administration both LAK generation and proliferation were enhanced when patients' PBMC were stimulated with IL-2 in vitro. However, when normal donors' PBMC were cultured in vitro in the presence Bryostatin and IL-2, LAK induction was inhibited while IL-2-driven proliferation was increased. These effects were also seen following only 2 h exposure to Bryostatin and could be elicited by conditioned medium from Bryostatin-pretreated cells. Neither IL-4 nor interferon was detected in the conditioned medium. Bryostatin in vitro was found to increase expression of IL-2 receptors on CD4⁺, CD8⁺ and CD56⁺ cells and augment the proportion of CD8⁺ cells in conjunction with IL-2. We conclude that Bryostatin in combination with IL-2 in vitro enhances proliferation and IL-2 receptor expression on lymphocytes, favouring CD8⁺ cells while suppressing the generation of LAK activity. Intravenous administration of Bryostatin increases the potential of IL-2 to induce proliferation and LAK activity in lymphocytes which, taken together with its putative direct antitumour effect, makes Bryostatin an interesting candidate for clinical trials in combination with IL-2.B.F. and P.L.S. are supported by the Cancer Research Campaign     

IL-6 enhances the cytotoxic activity of thymocyte-derived CD56+ cells.

Thymocyte-derived lymphokine-activated killer (LAK) cells were used as a model for the study of the cytokine driven development of cytotoxicity. These cells are devoid of initial cytotoxic activity but upon culture in IL-2 they develop into cytotoxic effectors. The parameters of the response of thymocytes to IL-6 are similar to that of PBL in that IL-6, at concentrations as low as 1 mu/ml, increases cytotoxicity of thymocyte-LAK cells when generated in low doses (25-50 mu/ml) of IL-2. IL-6-enhanced thymocyte-LAK cytotoxicity is observed when tested against both NK-resistant and NK-sensitive tumor cell lines. IL-6 alone does not induce any cytotoxicity from thymocytes nor does IL-6 change the time course of thymocyte-LAK cell generation in IL-2 culture. IL-6 does not affect DNA synthesis, total cell number, proportion of CD56+ cells, or the expression of IL-2R (both P55 and P75 glycoproteins) in IL-2-cultured thymocytes. Instead, IL-6 used to treat mature thymocyte-LAK effector cells for as little as 1 hr prior to 51Cr-release assay increases LAK cytotoxicity. This enhancement is abrogated by pretreatment of effector cells with cycloheximide, suggesting that protein synthesis is required for IL-6 to enhance LAK cell activity. The precursor phenotypes of IL-6-responsive thymocyte-LAK cells are CD3-/CD5-. The effector phenotypes of IL-6-enhanced thymocyte-LAK cells are CD5-/CD56+. Thus, IL-6 depends on synthesis of rapid-turnover proteins to act on mature CD56+/CD5- LAK cells to increase their cytotoxic function.     

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.     

Granulocyte-monocyte colony-stimulating-factor augments the interleukin-2-induced cytotoxic activity of human lymphocytes in the absence and presence of mouse or chimeric monoclonal antibodies (mAb 17-1A)

Summary Blood lymphocytes stimulated for 96 h with interleukin-2 (IL-2; 100 BRMP U/ml) (lymphokine-activated killer, LAK, cells) or granulocyte-monocyte colonystimulating-factor (GM-CSF) (10 ng/ml) became cytotoxic for Daudi cells. IL-2 was significantly more effective than GM-CSF. Only IL-2-activated cells killed SW948 (a human colorectal carcinoma cell line) while GM-CSF-stimulated cell did not. GM-CSF and IL-2 acted synergistically in a dose-dependent fashion for induction of a highly effective cytotoxic cell population (IL-2/GM-CSF cells). Il-2/GM-CSF cells were statistically significantly more effective than LAK cells in lysing Daudi cells and SW948 (P <0.05). The enhancing effect was most pronounced during the first 48–96 h of activation. Incubation periods longer than 192 h did not contribute to augmented cytotoxicity. The combination of IL-2 and GM-CSF significantly increased the number of CD25⁺ cells compared to IL-2 and GM-CSF alone. Furthermore, IL-2/GM-CSF cells were significantly more effective in antibody-dependent cellular cytotoxicity assays (SW948 + mAb 17-1A) than LAK cells. The chimeric mAb 17-1A was significantly more effective in tumor cell lysis than the mouse mAb. Thus, combination of various biological therapeutics might be a way to enhance their antitumoral effects.     

Generation of lymphokine-activated killer cell activity by low-dose recombinant interleukin-2 and tumor cells

The generation of lymphokine-activated killer (LAK) cells in vitro has been reported to require 100-1000 units of recombinant interleukin-2 (IL2). In this study we investigated the generation of human LAK cells with low-dose IL2 (1-10 U) in combination with human tumor cell lines. A significant LAK activity was generated within 3- to 5-days culture of PBL. Among six human tumor cell lines tested, the K562 cell line had the greatest stimulating activity, and the degree of cytotoxicity was comparative to that of PBL stimulated with higher doses of IL2 alone. The origin of this LAK activity was primarily the E(-) rosetting cell population. Cocultures of E- cells with 1 U/ml IL2 plus K562 had significantly higher cytotoxicity (P less than 0.05) compared to using E+ cells. Phenotypic analysis indicated that 1 U/ml IL2 plus K562 cell stimulation enhanced CD56+ and CD16+ cells. These studies suggest that very low dosages of IL2 with stimulator tumor cells can generate LAK activity comparable to that generated with high dosages of IL2 alone.     

IL-1 synergy with IL-2 in the generation of lymphokine activated killer cells is mediated by TNF-alpha and beta (lymphotoxin).

Interleukin 1 is a pleuripotent cytokine shown to synergize with IL-2 in the generation of lymphokine-activated killer (LAK) cells, when cultured with human peripheral blood mononuclear cells (PBMC) or peripheral blood lymphocytes (PBL). When IL-1 and low dose IL-2 are added in combination, both LAK cytotoxicity and proliferation are increased in short-term (5-6 day) and long-term (12-14 day) cultures compared with cells activated with IL-2 alone. The purpose of this study was to examine the contribution of tumor necrosis factor (TNF-alpha), lymphotoxin (LT, or TNF-beta) and the TNF receptor in the observed IL-1/IL-2 mediated synergy. Analysis of lymphocyte culture supernatants using the L929 bioassay and by specific ELISAs demonstrated an increased production of both TNF and LT in those cells cultured with IL-1 and IL-2. Utilizing specific neutralizing antisera, our experiments demonstrated the biologic activity of both cytokines, with LT-specific antibodies producing the greatest diminution of IL-1/IL-2 stimulated cell proliferation and cytotoxicity. The addition of IL-1 and IL-2 in combination markedly upregulated TNF-receptor expression (measured by Scatchard analysis) in comparison with cells stimulated with IL-2 alone. Characterization of the TNF-R by flow cytometric analysis revealed increased membrane expression of the 75 kDa, but not the 55 kDa, TNF binding protein as a result of IL-1 costimulation.     

IL-7 induces human lymphokine-activated killer cell activity and is regulated by IL-4.

Induction of lymphokine-activated killer (LAK) activity by IL-2 has been described and characterized as broadly cytolytic activity against both fresh and cultured tumors. rIL-7 in the absence of IL-2 also induces LAK activity in human cells. This activity is unique for IL-7, because it is not shared by other cytokines including IL-1, IL-4, IL-6, and TNF-alpha. IL-7 also induces either de novo or increased expression of the surface markers CD25 (Tac, IL-2R alpha-chain), CD54 (ICAM-1), Mic beta 1 (IL-2R beta-chain) and CD69 (early T cell activation Ag). IL-7-induced LAK activity is independent of IL-2 secretion, because it is not abrogated by IL-2 antisera. The LAK precursor responding to IL-7 stimulation is enriched in the null cell fraction as has been demonstrated for IL-2-induced LAK cells. TGF-beta and IL-4 interfere with generation of LAK activity by IL-7. Anti-IL-4 antiserum enhances IL-7-induced LAK activity and augments induction of surface marker expression by IL-7. This may be indirect evidence that IL-7 stimulation leads to induction of IL-4 activity. Our results describe the activation of mature lymphoid cells by IL-7. This and the previously described role of IL-7 in lymphohemopoiesis makes it a cytokine of potential therapeutic value for treatment of immunodeficiency states and possibly the immunotherapy of cancer.     

Effects of IL-7 and IL-2 on highly enriched CD56+ natural killer cells. A comparative study.

IL-7 has been shown to induce low levels of lymphokine-activated killer cell (LAK) activity in bulk PBMC populations. We report here that immunomagnetically purified CD56+ cells from peripheral blood generated high LAK activity in response to IL-7. The LAK activity induced by IL-7 was comparable to, or slightly lower than, the LAK activity induced by IL-2. When analyzing cells from the same donor, no detectable LAK-generating effect of IL-7 was registered in the PBMC population, in contrast to a substantial effect in the CD56+ population. IL-2 induced 8- to 15-fold higher proliferative activity in CD56+ cells, relative to IL-7. At suboptimal concentrations of IL-2, IL-7 had a synergistic effect on the proliferation. IL-2-neutralizing antibodies did not abrogate the IL-7-induced proliferation or LAK generation. Both IL-7 and IL-2 induced comparable levels of 75-kDa TNFR expression, whereas IL-2R alpha expression was higher in IL-7-stimulated CD56+ cells. Low levels of TNF were produced in response to IL-7 at day 5, as opposed to a 50-fold higher TNF production in response to IL-2. No IL-2 or IL-6 production was detected. Our data indicate that IL-7 has profound and direct effects on CD56+ cells.     

Human monocytes inhibit lymphokine-activated killer cell expansion in vitro

Depleting monocytes from human peripheral blood mononuclear cells (PBMC) enhances the in vitro activation of lymphokine-activated killer (LAK) cells. To determine if monocytes also altered LAK-cell expansion, we evaluated two methods of depleting monocytes from PBMC: nylon wool adherence (NWA) and phenylalanine methyl ester (PME) treatment. Both methods of depleting monocytes enhanced interleukin-2 (IL-2) driven, LAK-cell expansion; LAK expansion, however, was significantly greater after depletion with NWA than after PME. LAK cytotoxicity after NWA and PME depletion was equivalent. The degree of monocyte depletion, determined by evaluating morphology and the number of Leu-M3 (CD14) positive cells, and the proliferation of Leu 19 (CD56), OKT-3 (CD3), Leu2 (CD8), and Leu 3a (CD4) positive cells was also equivalent. Exposure of IL-2 activated cells to PME did not alter their cytotoxic activity. However, sequential treatment of PBMC with NWA, then PME, or with PME and then NWA, resulted in reduced expansion. This reduction in expansion was similar to PBMC treated with PME alone. Exposure of PME-depleted cells to nylon wool or to supernatants obtained from cells adherent to nylon wool further decreased LAK expansion relative to cells treated with NWA alone. We conclude that even at relatively low cell density, human monocytes markedly inhibit LAK-cell expansion in IL-2 driven PBMC cultures. Further, depletion of monocytes by NWA adherence is more effective than by treatment with PME, possibly due to subtle cellular damage induced by this latter treatment. These findings have implication for the in vitro and in vivo generation of LAK-cells by IL-2.     

Interleukin-7 mediates the generation and expansion of murine allosensitized and antitumor CTL.

Interleukin-7 (IL-7) is a 25-kDa cytokine that was initially described as a pre-B cell growth factor and more recently has been shown to cause T cell proliferation. We have investigated the in vitro effects of IL-7 on mature T cells to include the generation and further expansion of allospecific and antitumor CTL. B6 anti-DBA allospecific CTL were generated in the presence of IL-7, IL-2, the combination IL-7 plus IL-2, or no cytokine. IL-7 alone or when combined with IL-2 enhanced the generation of allospecific CTL. To evaluate the proliferative effects of IL-7, 4-day B6 anti-DBA cultures were cultured in IL-7, IL-2, or no cytokine. Cell proliferation and duration of growth of cells cultured in IL-7 were significantly greater than cells cultured in IL-2 or in the absence of cytokine. Allospecific cytolytic activity was maintained during proliferation in IL-7 to a maximum of 60 days. In contrast with the ability of IL-2 to generate LAK cells, murine splenocytes cultured at varying doses of IL-7 (1 to 10,000 ng/ml), resulted in minimal LAK cell activity. The effect of IL-7 in the generation of CTL with antitumor activity was also studied. Seven days after footpad injection of MCA 203 or 205 sarcoma, draining lymph nodes (DLN) were harvested and restimulated in vitro with MCA 203 or 205, respectively, and maintained in culture with either IL-7, IL-2, the combination of IL-7 plus IL-2, or no cytokine. After 10 days in culture, cells generated in IL-7 or IL-2 exhibited similar cytotoxicity against the syngeneic autologous MCA tumor. IL-7 generated cells, however, showed specificity when tested by 51Cr release which was not seen with IL-2-generated cells. Cells generated in IL-7 plus IL-2 were more cytolytic than cells cultured with either cytokine alone. To further define the mechanism of action of IL-7 in antitumor CTL cultures, a monoclonal antibody, S4B6.1, capable of blocking murine-specific IL-2 was employed. The partial inhibition by this mAb of the generation of antitumor CTL demonstrated that IL-7 acted, in part, by an IL-2-dependent mechanism. Finally, IL-7 cultures restimulated at Day 11 with autologous MCA 203 showed greater proliferation than IL-2 cultures and remained lytic at Day 21 of culture.(ABSTRACT TRUNCATED AT 400 WORDS)     

Partial correction of defective generation of lymphokine-activated killer cells in patients with chronic myelogenous leukaemia after in vivo treatment with interferon-α (wellferon)

Summary Patients with chronic myelogenous leukaemia (CML) in untreated chronic phase are deficient in their ability to generate lymphokine-activated killer (LAK) cells from peripheral blood mononuclear cells although they posses essentially normal levels of CD16⁺ and Leu19⁺ lymphocytes, which do not seem to be actively suppressed by tumour cells. Attempts to enhance LAK cell generation in these patients are reported here. Combining the lymphokines interleukins-2, with -4 and -5 (IL-2, IL-4, IL-5), was not successful; in fact, IL-4 depressed LAK cell induction in both normal donors and CML patients. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate also failed to enhance cytotoxicity of normal donors or patients, and indomethacin was similarly without effect. The only agent found to enhance LAK cell induction by IL-2 in normal donors was interferon- (but not IFN-) and even this modest effect was not seen with the cells of CML patients. Increasing concentrations of IL-2 and/or culture duration also failed to improve LAK cell generation by patients. The only improvement in LAK cell generation was observed in CML patients treated for one or more months with IFN-, where a steady increase of LAK activity with time after initiation of therapy was noted. These results show that the blockade of LAK cell induction in chronic-phase myelogenous leukaemia patients is difficult to lift pharmacologically in vitro but possibly susceptible to biological response modifiers in vivo.     

Clonal analysis of peripheral blood lymphocytes from three patients with advanced neuroblastoma receiving recombinant interleukin-2 and interferon α

In this study we have investigated, at the population and the clonal levels, the immunophenotypes and the non-specific cytotoxic functions of peripheral blood lymphocytes from three stage IV neuroblastoma patients receiving treatment with recombinant interleukin-2 (IL-2) and interferon (IFN). Both IL-2 alone and the combination of IL-2 and IFN caused an in vivo expansion of CD56⁺, CD3^– NK cells most of which expressed the p75 molecule, i.e. the chain of the IL-2 receptor. Peripheral blood mononuclear cells (PBMC), drawn after treatment, displayed an increased NK activity, but no lymphokine-activated killer (LAK) activity. However, the subsequent in vitro culture of PBMC with high-dose IL-2 induced the generation of a potent LAK activity, which was mediated by an expanded population of CD3⁺ CD8⁺ T cells. Finally lymphocytes that had been isolated after cytokine therapy were cloned, in the presence of low-dose phytohemagglutin, immediately or following culture with IL-2. Clones derived from LAK cells expanded in vitro had predominantly a CD3⁺, CD8⁺ immunophenotype, whereas those raised from freshly separated lymphocytes were either CD3⁺, CD4⁺ or CD3⁺, CD8⁺ in equal proportions. Most of the above clones were poorly or not at all cytolytic against NK-sensitive or NK-resistant targets. In contrast, the few NK clones obtained (CD3^–, CD56⁺) lysed all targets with high efficiency.This work was supported by a grant from Associazione Italiana per la Ricerca sul Cancro, Milano, Italy to V. P.     

IL-4 regulation of murine lymphokine-activated killer activity in vitro. Effects on the IL-2-induced expansion, cytotoxicity, and phenotype of lymphokine-activated killer effectors

The in vitro incubation of B6 splenocytes with purified, mouse rIL-4 for 4 to 5 days was sufficient to generate lymphokine-activated killer (LAK) activity. In addition, rIL-4 augmented LAK cytotoxic activity when combined with rIL-2, as measured in a 4 h 51Cr-release assay against fresh, syngeneic MCA-sarcoma (MCA-102 and MCA-105) cells. Interestingly, this augmentation was not observed against the cultured YAC-1 target. LAK generation and augmentation of cytotoxicity by rIL-4 was species-specific, because human rIL-4 (up to 20,000 U/ml) failed to elicit these effects in the mouse splenocyte cultures. When 5-day B6 LAK cells (splenocytes incubated in rIL-2 at 1000 U/ml for 5 days) were split and recultured in the combination of rIL-2 plus rIL-4 for 4 additional days at least a twofold greater expansion in cell number resulted compared to similar cells cultured in either rIL-2 or rIL-4 alone. Moreover, LAK cells expanded in rIL-2 plus rIL-4 exhibited substantial increases in in vitro cytolytic activity (on a per cell basis) against MCA-102 and MCA-105 sarcoma cells, but not against YAC-1 targets. FACS analysis or negative selection using Lyt-2 or NK-1.1 mAb plus C revealed no differences in effector phenotype(s) of LAK cells expanded in rIL-2 alone compared to rIL-2 plus rIL-4 to account for the differences observed in both expansion and cytolytic activity by rIL-4. The majority of cells was Thy-1+, Lyt-2+, T3+, and ASGM-1+. However, a marked increase in the granule-associated serine esterase, BLT-E, was found only in LAK cells expanded in the combination of both lymphokines. Collectively, these studies show that rIL-4 has potent regulatory activities on splenic LAK generation, expansion, and cytotoxic function in the mouse.     

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)