Induction Of Lymphokine-activated Killer Activity Research Articles

Combination chemo-immunotherapy: kinetics of in vivo and in vitro generation of natural killer cells and lymphokine-activated killer cells in the rat

Rats received a single high dose of cyclophosphamide (Cy) (150 mg/kg), followed 48 h later (on day 0) by immunization with a T cell-dependent soluble antigen, ovalbumin in Freund's complete adjuvant (FCA). The effect of this treatment on lymphoid cell subpopulations in the spleen, natural killer (NK) cell and interleukin-2 (IL-2) induced lymphokine-activated killer (LAK) cell activity was examined. Cy (with and without ovalbumin) caused a large relative increase (by day 14) in splenic OX8+, OX19- cells with NK morphology. A marked relative increase in fresh NK cell activity was noted after Cy + ovalbumin, but not consistently after Cy alone. Elevated NK activity was Cy dose- and time-dependent, was evident within 7 days post Cy/ovalbumin and persisted for at least 28 days. Pooled splenic mononuclear cells (MNC), obtained 14 days after Cy/ovalbumin, lost all cytolytic activity against YAC-1 cells when cultured in the absence of human recombinant IL-2 (rIL-2). In contrast, similarly maintained cells from normal rats displayed NK activity higher than normal 'fresh' levels. Upon culture in medium containing 500 U/ml rIL-2, however, 'augmented' NK activity was equivalent, on a per-cell basis, in both normal and Cy/ovalbumin-pretreated groups. LAK activity generated in vitro (i.e. against NK-resistant target cells) was significantly lower in the latter group, and the overall yield of cells was reduced. By day 21 after Cy/ovalbumin, augmented NK activity was significantly greater than controls, on a per-cell and total culture yield basis. Moreover, LAK activity was now similar between groups. It is concluded that the chemotherapy/immunization protocol which we have used can greatly enhance NK activity in vivo and that these cells are responsive to induction of LAK activity by IL-2 in vitro.

Interleukin-2 in neuroblastoma: clinical perspectives based on biological studies.

Stage IV neuroblastoma (NB) is a disease with a poor prognosis. Chemotherapeutical intensification and hematological rescue with autologous bone marrow transplantation (ABMT) achieve some complete remissions (CR), but most patients relapse during the first year. Immunotherapy could be an alternative in this situation of high risk of relapse due to residual disease and ABMT-related immunodepression. Ten stage IV NB patients in CR or very good partial remission have been treated with recurrent 5-day cycles of high doses of Interleukin-2 (IL2) after ABMT throughout one year (usually 5-6 cycles). Natural killer (NK) and lymphokine-activated killer (LAK) cytotoxic activities, as well as phenotype and number of circulating NK cells were determined, before and after each course of IL2 treatment. The effects promoted by IL2 varied during treatment: early cycles of IL2 induced a great extent of cell expansion, mainly on CD3-/CD16-/CD56+bright and CD8+dim cell phenotypes; conversely, late courses of IL2 promoted higher NK cytotoxic activity but a lesser increase on circulating NK cells. The induction of LAK activity did not significantly differ from early and late IL2 treatments. Clinical results are still inconclusive due to the small number of patients. The median follow-up of patients treated with IL2 is 24 months and the disease free survival (DFS) probability is 0.80 +/- 0.12 vs 0.16 +/- 0.15 from a historical control with identical treatment, but in the absence of IL2 treatment (p < 0.005). IL2 treatment-related toxicity was mild and no interruption of the treatment was required. Extremely accurate hydric control was carried out to avoid, as much as possible, the consequences of vascular leak syndrome, one of the most important toxic effects of IL2 treatment. The results presented here suggest an evolution of NK activity during IL2 treatment after ABMT, which should be taken into account for the designing of new immunotherapeutical protocols and opens a promising perspective in treatment of stage IV neuroblastoma.

Induction of lymphokine-activated killer activity in mice by prothymosin alpha.

We have recently demonstrated that prothymosin alpha (ProT alpha) when administered intraperitoneally (i.p.) protects DBA/2 mice against the growth of syngeneic leukemic L1210 cells through the induction of tumoricidal peritoneal cells producing high levels of tumor necrosis factor alpha (TNF alpha) [Papanastasiou et al. (1992) Cancer Immunol Immunother 35: 145]. In this report we tested further immunological alterations that may be caused by the administration of ProT alpha in vivo. We demonstrate that i.p. injections of ProT alpha enhance natural killer (NK) cell activity and induce lymphokine-activated (LAK) activity in vivo. Thus, splenocytes from ProT alpha-treated DBA/2 animals exhibited significantly higher cytotoxic activity (up to threefold) against the NK-sensitive YAC cell line and the NK-resistant P815 and L1210 syngeneic tumor cells, as compared to splenocytes from syngeneic control mice. The enhancement of the cytotoxic profile of DBA/2 splenocytes was associated with increased percentages of CD8+ cells, NK cells and activated CD3+ cells. The ProT alpha-induced effect persisted for 30 days after the end of the ProT alpha treatment period and returned to normal levels 20 days later. Splenocytes from non-treated DBA/2 animals generated high NK and LAK activities in response to ProT alpha in vitro. The ProT alpha-induced NK and LAK activities reached 84% and 75% respectively of what was obtained with interleukin-2 (IL-2). High concentrations of TNF alpha and IL-2 were generated in response to ProT alpha in LAK cultures. These findings suggest that ProT alpha may provide an overall protective effect against tumor growth in vivo through induction of NK and LAK activities possibly indirectly via the production of IL-2 and TNF alpha in the spleen, peritoneal cavity and probably other lymphoid organs.

Enhanced induction of lymphokine-activated killer activity following a single dose of cisplatin in cancer patients

The effects of intravenous cisplatin (CDDP) administration on the generation of lymphokine-activated killer (LAK) activity in peripheral blood mononuclear (PBM) cells were investigated in cancer patients. The ability of PBM to generate LAK activity was significantly augmented 3, 5 and 7 days after a single dose, 50 mg m −, of CDDP injection when compared to that before injection. NK activity of PBM was not altered. The distribution of lymphocyte subsets exhibited no significant change following CDDP injection, except CD2 + cells. However, the ability of monocytes in PBM to produce TNF-α was significantly enhanced 5 days after the drug administration, although 1L1-β production was not augmented.

Induction of lymphokine-activated killer activity in mice by prothymosin ?

Induction of lymphokine-activated killer activity in mice by prothymosin ?

Requirement of thiol compounds as reducing agents for IL-2-mediated induction of LAK activity and proliferation of human NK cells.

Thiol-related compounds, such as L-cystine, 2-ME or reduced glutathione (GSH), are important in many lymphoid cell activation pathways. We investigated their role in IL-2-generated lymphokine-activated killer (LAK) activity in human NK (CD16+ and/or CD56+) cells. Depletion of GSH and L-cystine, but not L-leucine or glycine, from medium used during culture of NK cells with IL-2 inhibited LAK and proliferative activities, whereas IL-2-independent lytic function of NK cells remained intact. Addition of L-cysteine, 2-ME or GSH, but not methylated analogs of these compounds (which cannot function as proton donors) to L-cystine/GSH-depleted medium restored proliferative response of NK cells and LAK generation. In the presence of L-buthionine-(S,R)-sulfoximine, an inhibitor of GSH synthesis, IL-2-induced LAK activity and proliferation of NK cells in medium without L-cystine and GSH, could be restored, at least in part, by addition of GSH, but not 2-ME or L-cystine. Furthermore, intracellular GSH levels were depressed in cells cultured in L-cystine/GSH-depleted medium but could be restored by the addition of 2-ME. The results suggest that (1) L-cystine or thiol containing compounds such as L-cysteine, 2-ME, or GSH are necessary for effective IL-2-activation of human NK cells, (2) these compounds must be functional proton-donors, i.e., reducing agents, implying regulation of the IL-2 activation pathway by oxidation-reduction, and (3) GSH synthesis is necessary for the activation. Experiments were performed to begin to dissect the redox-sensitive step(s) in LAK development. Depletion of reducing agents had no effect on internalization of rIL-2. In contrast, intracellular granzyme A activity was significantly depressed in NK cells cultured with rIL-2 in L-cystine/GSH-depleted medium compared with those cultured in medium in which L-cystine levels had been replenished. The findings suggest that step(s) in the transduction of the IL-2 signal in NK cells, between the internalization of IL-2 and the maturation of the lytic mechanism, are subject to regulation by oxidation-reduction.

Phenotypic and functional profile of peripheral blood mononuclear cells isolated from melanoma patients undergoing combined immunotherapy and chemotherapy.

In the present study we tested the phenotypic profile as well as several immunological responses of peripheral blood mononuclear cells (PBMC) isolated from melanoma patients. These patients underwent chemotherapy with dacarbazine and carboplatin from day 1 to day 22, followed by immunotherapy of low-dose recombinant interleukin-2 and recombinant interferon alpha administered subcutaneously from day 36 to day 75. The PBMC from 14 patients were isolated on day 0 before chemotherapy, on day 36 after chemotherapy and on day 76 after immunotherapy. After chemotherapy, a decrease in CD16+ cells and increase in CD3+ and CD4+ cells correlated with a significant decrease in the generation of lymphokine-activated killer (LAK) activity. After immunotherapy, an increase in CD16+ cells correlated with an increase in the induction of LAK activity. A comparison between responding and non-responding patients revealed statistically significant differences in LAK activity of PBMC and response to concanavalin A following chemotherapy, and in the percentage of CD8+ cells following immunotherapy. Our results point toward the value of continuing such a study on a larger population of cancer patients in order to select the appropriate bioassays for monitoring and predicting the clinical responsiveness to combined therapies.

Enhanced induction of lymphokine-activated killer activity after lentinan administration in patients with gastric carcinoma

In 15 patients with gastric carcinoma, peripheral blood mononuclear cells (PBM) were obtained serially before and 3, 5 and 7 days after lentinan administration. The generation of lymphokine-activated killer (LAK) activity, induced by in vitro activation of PBM with interleukin 2 (IL 2), was significantly augmented 5 days after a single intravenous dose of 2 mg lentinan, when compared with that before lentinan injection. Natural killer (NK) activity of PBM was also significantly enhanced 7 days after the drug injection. However, the distribution of lymphocyte subsets exhibited no significant change following lentinan administration.

Induction of lymphokine-activated killer activity in rat splenocyte cultures: the importance of 2-mercaptoethanol and indomethacin.

The role of 2-mercaptoethanol and indomethacin in the induction of lymphokine-activated killer (LAK) activity by interleukin-2 (IL-2) in rat splenocyte cultures was investigated. Spleens from 4-month-old male rats of five different strains were tested. Splenocytes were cultured for 3-5 days in the presence of IL-2 (1000 U/ml) and LAK activity was assessed by 4-h 51Cr release assays with P815 and YAC-1 cells as targets. LAK activity could be induced by IL-2 in splenocytes from all rat strains, but only when 2-mercaptoethanol was present in the culture medium. Optimal LAK activity was induced when the 2-mercaptoethanol concentration in splenocyte cultures was at least 5 microM. Different rat strains showed differences in levels of in vitro induction of LAK activity. In the presence of 2-mercaptoethanol the level of LAK activity induced by IL-2 was high in BN and Lewis rats, intermediate in Wistar and Wag rats, and low in DZB rats. In the absence of 2-mercaptoethanol no or minimal LAK activity was induced. Furthermore we observed that addition of 50 microns indomethacin to the culture medium in the presence of 2-mercaptoethanol augmented the induction of LAK activity to some extent. In the absence of 2-mercaptoethanol, addition of indomethacin resulted only in low levels or no induction of LAK activity. We conclude that for optimal induction of LAK activity by IL-2 in rat splenocyte cultures 2-mercaptoethanol is essential, while indomethacin can only marginally further improve this induction.

Prolonged Interleukin-2 (IL-2) Treatment Can Augment Immune Activation Without Enhancing Antitumor Activity in Renal Cell Carcinom

Preliminary studies involving small numbers of patients have suggested that interleukin-2 (IL-2) administered by continuous infusion in repetitive weekly cycles using doses of 3 x 10(6) U/M2/day is immunologically active and can induce tumor responses in patients with renal cell carcinoma. This study was designed to examine both the immunological and clinical effects of prolonged infusion IL-2 given by repetitive weekly cycles; first at moderate doses for 4 weeks as an impatient followed by lower doses of IL-2 for up to 5 months. Prolonged IL-2 treatment was investigated because previous studies revealed that patients had a return to their baseline immune status within 4 weeks after completing IL-2 treatment. Twenty-five patients (including 18 with renal cell carcinoma) were treated with one of two regimens utilizing IL-2 as sole therapy. These regimens were designed to induce augmented and prolonged immune activation based upon in vitro and in vivo data. Though patients on both arms of the study demonstrated sustained lymphocytosis, increase in numbers of natural killer cells, and induction of lymphokine-activated killer activity with prolonged IL-2 administration, only 1 out of the 18 patients with renal cell carcinoma demonstrated a sustained partial antitumor response to therapy. Furthermore, several patients demonstrated profound immune activation, without any evidence of tumor regression. The lack of clinical responses in these patients showing marked activation of LAK cytotoxicity suggests that other variables must also influence the likelihood of antitumor effects for patients receiving IL-2 therapy.

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.

Lymphokine activated killer (LAK) activity in normal pregnancy

With a view to define inherent tendency in activity of lymphokine activated killer (LAK) cell during pregnancy a series of test has been conducted with consequently evolved results quoted in the following summary;1) In an extensive program of test there was a marked fall in activity of LAK significantly over the period from initial-to third trimester (p<0.01). It, however, rapidly recovered at the occasion of delivery until consequent difference has become no longer distinctive when compared to the equivalent of non-pregnant subject.2) Another research directed to a possible influence of adherent cells and pregnant serum on the LAK activity which might possibly account for the loss of LAK activity, gave a conclusive evidence to support that both factors would give none of influence on the LAK activity.3) Further study through flow cytometry on the behavior of lymphocyte subsets (OKT3, OKT4, OKT8, Leu7, Leull, OKIal and OKM1) under cultivation made with added interleukin 2 (IL-2) did show none of difference between pregnant-and non-pregnant subjects. From this fact it may be retrospectively inferred that loss of LAK activity during pregnancy would be attributed to dysfunction rather than to reduced number of LAK cells.4) A factor to suppress IL-2 production capability of peripheral blood lymphocytes under PHA stimulation was identified in the serum of pregnant subjects, whereas it was not the case with the subjects during puerperal phase. With the above decisive fact it has been shown convincingly that induction of LAK activity during pregnancy would have been suppressed by etiological mechanism involving both loss of IL-2 production and dysfunction of LAK cells, viz. an immunological environment helpful to maintain pregnancy may have been thereby provided for.