Induction Of Lymphokine-activated Killer Cells Research Articles

Enhancement of lymphokine-activated killer cell induction using anti-CD25 and anti-CTLA-4 monoclonal antibodies

Immunosuppression may contribute to cancer progression, in which regulatory T (T-reg) cells have been demonstrated to play important roles. We investigated whether anti-CD25 (alpha-CD25) monoclonal antibody (mAb) and anti-cytotoxic T lymphocyte-associated antigen-4 (alpha-CTLA-4) mAb could augment in vitro proliferation and cytotoxic activity against cancer cell lines of lymphokine-activated killer (LAK) cells. Human LAK cells with immobilized alpha-CD3 Ab plus IL-2 were significantly augmented, including LAK/alpha-CD25 (10 microg ml, p=0.045) and LAK/alpha-CTLA-4 (5 microg/ml, p=0.025; 10 microg/ml, p=0.019). LAK/alpha-CD25 and LAK/alpha-CTLA-4 showed significant cytotoxic activities against gastric cancer cell lines (p<0.05). The phenotype of LAK cells showed that alpha-CD25 and alpha-CTLA-4 mAb more selectively induced the phenotype of CD8+ cells. The secretion of IFN-gamma increased significantly in LAK/alpha-CTLA-4 (p=0.032). alpha-CD25 mAb reduced intracellular CTLA-4 (p=0.0069), and alpha-CTLA-4 mAb reduced intracellular FOXP3 (p=0.049), respectively. These results suggest that LAK cells are highly augmented in the presence of alpha-CD25 mAb and alpha-CTLA-4 mAb through the possible mechanism of the suppression of T-reg.

Inhibition of Lymphokine-Activated Killer Cells Generation In Vitro by Soluble Factors Released from Mixed Human Tumor and Peripheral Blood Mononuclear Adherent Cells Culture

Background: A variety of molecules produced by both tumor cells and normal cells reduce the activity of lymphokine-activated killer (LAK) cells. We tested the possible cross-regulation of mel-624 melanoma cells and adherent peripheral blood mononuclear cells (PBMCs) in affecting LAK cell activity. Methods: PBMC adherent cells were cultured together with mel-624 melanoma cells. Supernatant was transferred to a 4-day LAK cells generation culture consisted of PBMC nonadherent cells and interleukin-2. LAK cytotoxic activity was tested in a 4-hour assay against Daudi tumor cells prelabeled with sodium 51chromate. Results: The supernatant produced within the first 48 hours of mixed mel-624 melanoma cell and adherent PBMC culture substantially (by 69 percent) reduced the generation of LAK cells, whereas the supernatant from either tumor culture or adherent PBMC culture had no effect. The inhibitory effect was manifested on the generation of LAK cells when autologous nonadherent cells were cultured with 1,000 units/ml IL-2, but there was no effect on mature LAK cell cytotoxic activity. Inhibition of LAK cell generation was partially dependent on protein synthesis and was not mediated by transforming growth factor β (TGF-β). Conclusion: Our results point toward the production of soluble, yet unidentified proteins, in mixed tumor-adherent PBMC cultures, which substantially reduced the induction of LAK cells in culture.

Activation of CD38 by Interleukin-8 Signaling Regulates Intracellular Ca2+ Level and Motility of Lymphokine-activated Killer Cells

CD38 is an ADP-ribosyl cyclase, producing a potent Ca(2+) mobilizer cyclic ADP-ribose (cADPR). In this study, we have investigated a role of CD38 and its regulation through interleukin-8 (IL8) signaling in lymphokine-activated killer (LAK) cells. Incubation of LAK cells with IL8 resulted in an increase of cellular cADPR level and a rapid rise of intracellular Ca(2+) concentration ([Ca(2+)](i)), which was sustained for a long period of time (>10 min). Preincubation of an antagonistic cADPR analog, 8-Br-cADPR (8-bromo-cyclic adenosine diphosphate ribose), abolished the sustained Ca(2+) signal only but not the initial Ca(2+) rise. An inositol 1,4,5-trisphosphate (IP(3)) receptor antagonist blocked both Ca(2+) signals. Interestingly, the sustained Ca(2+) rise was not observed in the absence of extracellular Ca(2+). Functional CD38-null (CD38(-)) LAK cells showed the initial rapid increase of [Ca(2+)](i) but not the sustained Ca(2+) rise in response to IL8 treatment. An increase of cellular cADPR level by cGMP analog, 8-pCPT-cGMP (8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate), but not cAMP analog or phorbol 12-myristate 13-acetate was observed. IL8 treatment resulted in the increase of cGMP level that was inhibited by the IP(3) receptor blocker but not a protein kinase C inhibitor. cGMP-mediated Ca(2+) rise was blocked by 8-Br-cADPR. In addition, IL8-mediated LAK cell migration was inhibited by 8-Br-cADPR and a protein kinase G inhibitor. Consistent with these observations, IL8-induced migration of CD38(-) LAK cells was not observed. However, direct application of cADPR or 8-pCPT-cGMP stimulated migration of CD38(-) cells. These results demonstrate that CD38 is stimulated by sequential activation of IL8 receptor, IP(3)-mediated Ca(2+) rise, and cGMP/protein kinase G and that CD38 plays an essential role in IL8-induced migration of LAK cells.

Nitric Oxide Exposure Inhibits Induction of Lymphokine-Activated Killer Cells by Inducing Precursor Apoptosis

Nitric oxide synthesis is strongly induced during IL-2 treatment of mice and humans. While this free radical can act as an antitumor mechanism by inhibiting cellular respiration and DNA synthesis in cancer cells, immunosuppressive effects have also been suggested. We evaluated the effects of NO exposure on the induction of murine lymphokine-activated killer (LAK) cells from splenocytes by IL-2 (6000 IU/ml). When splenocytes were exposed to pure NO gas for 30 min prior to the addition of IL-2, complete abrogation of LAK cell cytotoxicity was observed. In contrast, cytolytic activity of already activated LAK cells was only minimally affected by NO exposure. NO exposure markedly depressed cellular proliferation in response to concanavalin A or IL-2. Immunostaining of LAK cell cultures following NO exposure revealed a marked decrease in CD8+, and peanut lectin (PNA+)/CD56+subsets (48 and 69%). Dual staining of LAK cells for DNA strand breaks and either PNA or CD8+identified the induction of programmed cell death in these subsets 12–24 h following NO exposure. These experiments demonstrate that NO has the capacity to inhibit LAK cell induction by inducing apoptosis of cytolytic lymphocyte precursors.

IL-2 induces Fas ligand/Fas (CD95L/CD95) cytotoxicity in CD8+ and CD4+ T lymphocyte clones.

IL-2 is a T cell growth factor that has pleiotropic functions in T cell differentiation, induction of lymphokine-activated killer cells, and regulation of immune responses. In studying TCR triggering of perforin or Fas ligand (FasL)/Fas (CD95 ligand/CD95) cytotoxicity in our influenza-specific T cell clones, we found that IL-2 can also induce FasL/Fas cytotoxicity. IL-2 induces FasL/Fas cytotoxicity in our CD8+ and CD4+ Th1 clones, but not in our CD4+ Th2 clones. IL-2 induction of cytolytic activity occurs when the CD8+ T cells are refractory to IL-2-induced proliferation. This killing is Ag independent, MHC unrestricted, and blocked by Fas.Fc fusion protein. IL-2 induces FasL/Fas cytotoxicity in a dose-dependent manner, but does not induce high levels of FasL expression as detected by flow cytometry. TCR triggered FasL/Fas cytotoxicity is detectable in CD8+ and Th1 clones by 3 h and peaks at 6 h; high levels of killing are maintained for at least 24 h. Similarly, IL-2 induces FasL/Fas killing in CD8+ and Th1 clones within 3 h of stimulation and maintains high levels for at least 24 h. TCR-triggered FasL/Fas killing is inhibited by emetine and cyclosporin A, whereas IL-2-induced FasL/Fas killing is inhibited by emetine, but not by cyclosporin A. These results demonstrate a second mechanism to induce FasL/Fas cytotoxicity in CD8+ and Th1 clones and may explain IL-2 induction of Ag-independent MHC-unrestricted lymphokine-activated killer cell activity.

Quantitation of human IL-12 heterodimer and p40

Interleukin 12 (IL-12), formerly known as cytotoxic lymphocyte maturation factor and natural killer cell stimulatory factor, is a cytokine secreted by a human B lymphoblastoid (NC-37) cell line when induced in culture with phorbol ester and calcium ionophore. This factor has been purified to homogeneity and shown to synergize with low concentrations of interleukin 2 in causing the induction of lymphokine-activated killer cells. In addition, purified IL-12 stimulated the proliferation of human phytohemagglutinin-activated lymphoblasts by itself and exerted additive effects when used in combination with suboptimal amounts of interleukin 2. The protein is a heterodimer composed of a 40- and a 35-kDa subunit. Amino acid sequence analysis confirmed predicted sequences from the cloned cDNAs of each subunit. Chemical and enzymatic deglycosylation of the heterodimer demonstrated that the 40- and 35-kDa subunits contain 10 and 20% carbohydrate, respectively. Structural analysis of IL-12 using site-specific chemical modification revealed that intact disulfide bonds are essential for bioactivity. The 40-kDa subunit of IL-12 was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by immunoblotting as being present in NC-37 cell supernatant solutions in relatively large amounts uncomplexed to the 35-kDa subunit. Previously it had been shown that the 40-kDa subunit alone does not cause the proliferation of activated human T lymphocytes or enhance the cytolytic activity of human natural killer cells. However, results obtained by site-specific chemical modification suggesting that a tryptophan residue is at or near the active site of IL-12 may imply a direct role of the subunit in interacting with the IL-12 receptor. These data may support the recent proposal (D. P. Gearing and D. Cosman (1991)Cell 66, 9–10) that IL-12 consists of a complex of cytokine and soluble receptor.

Generation of primary tumor-specific CTL in vitro to immunogenic and poorly immunogenic mouse tumors.

This study investigated the generation of primary tumor-specific CTL activity in vitro to several mouse tumors. We report that the development of optimal primary tumor-specific CTL to the P815 mastocytoma, the EL4 thymoma, and the Lewis lung carcinoma is dependent on tumor Ags, on enhancement of T cell costimulation by B7.1, and on exogenous T helper activity in the form of IL-2 and IL-4. A relatively low concentration of IL-2 and IL-4 was required to limit the induction of lymphokine-activated killer cells. In the case of P815, the CTL were directed toward molecularly defined tumor rejection Ags. These primary cultures yielded long term T cell lines that were heterogeneous in fine tumor Ag specificity and in cytokine production.

Immune reactions induced by interleukin-2 transfected colorectal cancer cells in vitro: predominant induction of lymphokine-activated killer cells.

One aim of the genetic modification of tumor cells is the generation of immunogenic variants that can be used for the induction of immune responses against tumors. We engineered the human colorectal carcinoma cell line SW480 by means of plasmid transfection to secrete interleukin (IL)-2. Transfection of SW480 cells resulted in stable IL-2 secretion at 5-30 ng/ml per 10(5) cells in 24 h and, unexpectedly, in CD54 expression on the cell surface. SW480 variants expressing IL-2 and CD54 were tested for their capacity to induce T lymphocyte activation in vitro in comparison to untransfected and CD54 transfected cells. The cytolytic effector function of a class I MHC restricted CD8+, peptide antigen specific T cell clone was augmented following expression of CD54. IL-2 secreting SW480 variants did not further increase antigen-dependent cytolysis. Primary activation of resting T lymphocytes was assessed following allogeneic stimulation. When compared with unmodified SW480 cells, CD54 expressing variants did not initiate T cell proliferation. In contrast, IL-2 secreting SW480 cells strongly promoted primary T cell proliferation. Similarly, exogenous IL-2 and SW480 cells induced T cell proliferation which was not only due to IL-2 but was dependent on tumor cells. However, following the initial wave of cell growth in response to IL-2 secreting SW480 cells T lymphocytes could not be restimulated with SW480 or IL-2 secreting variants and could not be further expanded. T cells initially activated by IL-2 secreting SW480 cells exhibited cytolytic activity towards SW480 cells. This reactivity, however, was transient and completely blocked by K562 cells, suggesting MHC-unrestricted, nonspecific cytotoxicity. We conclude that endogenous IL-2 secretion by the colorectal carcinoma cell line SW480 does not result in the activation of MHC restricted specific T lymphocytes but predominantly induces lymphokine-activated killer cells. Considering that tumor cell vaccines are aimed at inducing tumor-specific immune responses, our in vitro observation would rather argue against the in vivo application of such a tumor cell modification in colorectal cancer.

Effectiveness and toxicity of protracted nitric oxide synthesis inhibition during IL-2 treatment of mice.

The current study was designed to characterize nitric oxide (NO.) synthesis during interleukin-2 (IL-2) treatment of mice, and to determine whether NO. mediated IL-2-induced "vascular leak." We developed a technique for chronic subcutaneous infusion of the NO. synthase inhibitor N omega monomethyl-L-arginine (MLA) via osmotic minipump to aid in further study of these processes. After IL-2 administration to C3H/HeN mice (180,000 IU i.p. b.i.d. for 5 days), NO. synthesis increased two-to-three fold, peaking on days 5-8. Administration of MLA reduced NO. synthesis in both IL-2-treated mice (from 2.7 to 1 microM/mouse/day), and normal mice (from 1 to 0.5 microM/mouse/day). This agent decreased IL-2-induced radiolabeled albumin accumulation in the liver after i.p. IL-2 administration (p < 0.02). MLA infusions resulted in minimal systemic toxicity in mice, as reflected by complete blood counts or serum chemistries. MLA also did not impair lymphokine-activated killer cell induction in vitro or in vivo, or alter IL-2-induced tumor responses in a 3-day pulmonary metastasis model. These experiments demonstrated that NO. is a mediator involved in the genesis of vascular permeability induced by IL-2 treatment. Studies designed to further evaluate the toxicity and usefulness of MLA infusions to modify this IL-2 induced toxicity appear to be warranted.

Interleukin-12.

Interleukin (IL)-12 was originally identified as a factor produced by human Epstein-Barr virus-transformed B cell lines. It was detected by one group as cytotoxic lymphocyte maturation factor, a cytokine that synergized with IL-2 in the induction of lymphokine-activated killer cells and cytotoxic T lymphocytes. A second group characterized it as a natural killer (NK) cell stimulatory factor, due to the enhancement of cytotoxicity and IFN-gamma synthesis by NK cells. Human IL-12 was purified to homogeneity and cloned by both groups. We had identified a murine factor, provisionally termed T cell-stimulating factor (TSF), which was involved in the proliferation, synthesis of IFN-gamma and cell adhesion of CD4+ Th1 cells. TSF was produced in the antigen-specific interaction between Th1 cells and macrophages as antigen-presenting cells, partially purified from supernatants of such cultures, and shown to be identical to IL-12. Monocytes/macrophages appear to be the major source of IL-12. It is rapidly produced by phagocytic cells after stimulation with several bacteria/bacterial products and other microorganisms. In the light of its effects on NK cells as well as CD4+ and CD8+ T cells, IL-12 can be regarded as a cytokine that connects the innate immune system with the acquired immunity. IL-12 has a broad range of activities already reviewed in three papers. These include the regulation of cytokine synthesis and proliferation of T and NK cells, the promotion of Th1 cell development, the differentiation of CD8+ T cells and effects on hematopoiesis. When applied in vivo, IL-12 was shown to enhance the resistance to bacterial and parasitic infections, to promote antitumor immunity, and to influence antiviral responses including HIV in vivo or in vitro. This review will briefly summarize these effects, but mainly focus on recent results concerning the regulation of the production and the activity of IL-12, its role in the differentiation of Th cells and the implications for delayed- and immediate-type hypersensitivity reactions, its importance for organ-specific autoimmune diseases, and the possible role of the IL-12p40 homodimer as a specific inhibitor of the IL-12 heterodimer.

A lymphokine, provisionally designated interleukin T and produced by a human adult T-cell leukemia line, stimulates T-cell proliferation and the induction of lymphokine-activated killer cells.

In early phases of human T-cell lymphotrophic virus I-induced adult T-cell leukemia (ATL), the malignant cell proliferation is associated with an autocrine process involving coordinate expression of interleukin (IL) 2 and its receptor. However, during late-phase ATL, leukemic cells no longer produce IL-2 yet continue to express high-affinity IL-2 receptors. During studies to define pathogenic mechanisms that underlie this IL-2-independent proliferation, we demonstrated that the ATL cell line HuT-102 secretes a lymphokine, provisionally designated IL-T, that stimulates T-cell proliferation and the induction of lymphokine-activated killer cells. Conditioned medium from HuT-102, when added to the IL-2-dependent CTLL-2 line, yielded a stimulation index of 230. Since CTLL-2 was purported to be IL-2-specific, we performed a number of studies to exclude IL-2 production by HuT-102. Stimulation of CTLL-2 cells by HuT-102-conditioned medium was not meaningfully inhibited by addition of an antiserum to IL-2. Furthermore, uninduced HuT-102 cells did not express mRNA encoding IL-2 as assessed by Northern blot analysis. No biological activity on CTLL-2 cells was mediated by purified IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, or granulocyte/macrophage colony-stimulating factor, thus differentiating these factors from IL-T. Based on preliminary biochemical data, IL-T is a protein with a pI value of 4.5 and a molecular mass in SDS gels of 14 kDa. In addition to its action on CTLL-2 cells, 3200-fold-purified IL-T stimulated proliferation of the human cytokine-dependent T-cell line Kit-225. Furthermore, addition of IL-T enhanced cytotoxic activity of large granular lymphocytes (i.e., induced lymphokine-activated killer cells). Thus, IL-T is a lymphokine that plays a role in T-cell proliferation and induction of lymphokine-activated killer cells. Furthermore, IL-T may contribute to IL-2-independent proliferation of select ATL cells and lines.

The interleukin (IL) 2 receptor beta chain is shared by IL-2 and a cytokine, provisionally designated IL-T, that stimulates T-cell proliferation and the induction of lymphokine-activated killer cells.

Late-phase human T-cell lymphotropic virus I-associated adult T-cell leukemia cells express IL-2 receptors (IL-2R) but no longer produce IL-2. We have reported that the IL-2-independent adult T-cell leukemia line HuT-102 secretes a cytokine, provisionally designated IL-T, that stimulates T-cell proliferation and lymphokine-activated killer cell activity. Stimulation of proliferation of the cytokine-dependent human T-cell line Kit-225 mediated by HuT-102-conditioned medium or by 3200-fold-purified IL-T was not blocked by the addition of antibodies against IL-2 or IL-2R alpha subunit. However, IL-T-mediated stimulation of this human T-cell line was inhibited by addition of Mik-beta 1, an antibody that binds specifically to IL-2R beta subunit. In addition, the activation of large granular lymphocytes to lymphokine-activated killer cells mediated by IL-T-containing conditioned medium was not blocked by antibodies directed toward IL-2 or IL-2 alpha but was inhibited by an antibody to IL-2R beta, suggesting the requirement of this receptor subunit for IL-T action. This conclusion was confirmed using an IL-3-dependent murine myeloid precursor cell line, 32D, that expresses IL-2R alpha and IL-2R gamma, but not IL-2R beta. Neither IL-2 nor IL-T stimulated 32D cell proliferation. However, after transfection with the gene encoding human IL-2R beta, 32D beta cells proliferated on addition of either cytokine. The IL-T-mediated stimulation of 32D beta proliferation was inhibited by an anti-IL-2R beta antibody but not by an anti-IL-2 antibody. Thus, the IL-T-mediated stimulation of T-cell and lymphokine-activated killer cell activation requires the expression of the IL-2R beta subunit.

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In order to reduce the inherent risk of serum-supplemented media in cancer patients treated with LAK cells and induce high cytotoxic activity, we have developed a serum-free medium designated as RD5F. RD5F medium consists of transferrin, 2-aminoethanol, 2-mercaptoethanol, sodium selenite and interleukin-2 in addition to nutrient mixture. The cytotoxic activity of LAK cells generated in RD5F is higher than that in medium containing 10% human AB serum against Raji, K562 cells, and oral cancer cells. We have also found that insulin and insulin-like growth factor-1 (IGF-1), which is considered to be the most important hormone of serum-free media for animal cells, inhibited the induction of cytotoxic activity of LAK cells derived from PBL and RLNL, and that transferrin was an essential component of serum-free media for induction of LAK cells. The results obtained by two-color FACS analysis suggested that insulin stimulated differentiation of PBL to CD4+ cells in serum-free culture. Tumor infiltrating lymphocytes (TIL) generated in RD5F medium exhibited CD3 + HLA-DR + phenotype and much higher cytotoxicity against autologous tumor cells than that against allogenic cells. These results strongly suggest the possibility that insulin and IGF-1 act as immunosuppresive factors. In addition, RD5F medium was suggested to be useful in the adoptive immunotherapy of cancer.

Experimental study on interleukin 6 activity against tumor; Regulation of interleukin-2 induced lak cell activity

The ability of human recombinant interleukin-6 (IL-6) to regulate the induction and cytotoxic function of lymphokine activated killer (LAK) cells from human fetal spleen was studied. The results showed that IL-6 alone was unably to induce fetal splenic mononuclear cells (FSMC) to develop functional LAK cells, nor was it able to affect the number of IL- 2- induced LAK cells and to alter the lytic activity of LAK cells against tumor cells in effector phase. However, when IL- 6 was used in combination with IL- 2 during the induction phase, the resulting LAK cells displayed considerably greater lytic, activity than that with IL-2 alone (P<0.01), and this effect was IL-6 dose-dependent. The possible machanism of the synergistic effect of IL-2 and IL-6 in LAK cell induction from human fetal spleen is discussed.

Effect of Granulocyte-Macrophage Colony-Stimulating Factor on Lymphokine-Activated Killer Cell Induction

AbstractThe treatment of cancer with lymphokine-activated killer (LAK) cells in conjunction with high-dose interleukin-2 (IL-2) has been limited by the toxicity of IL-2 and the narrow range of tumors that respond to therapy. Cytokines that are capable of augmenting lower doses of IL-2 are, therefore, a major focus of research. We report here that granulocyte-macrophage colony-stimulating factor (GM-CSF) can augment low-dose IL-2 LAK induction from murine splenocytes. Anti-tumor necrosis factor α (anti-TNFα) or anti-interferon γ (anti-IFNγ) monoclonal antibodies did not inhibit (IL-2 + GM-CSF)-induced LAK generation, indicating that GM-CSF augmentation does not require TNFα or IFNγ activity. Depletion of natural killer cells before culture did not inhibit low-dose IL-2–induced LAK generation or the ability of GM-CSF to augment LAK generation. In contrast, depletion of both CD4+ and CD8+ T cells before culture inhibited the generation of LAK activity. However, depletion of only CD4+ T cells, or only CD8+ T cells, did not inhibit the generation of IL-2 or (IL-2 + GM-CSF) LAK activity. These results suggest that LAK precursors are present in both the CD4+ and CD8+ T-cell populations and suggest that the addition of GM-CSF to low-dose IL-2 may result in the generation of T-derived LAK cells.

Effect of granulocyte-macrophage colony-stimulating factor on lymphokine-activated killer cell induction

The treatment of cancer with lymphokine-activated killer (LAK) cells in conjunction with high-dose interleukin-2 (IL-2) has been limited by the toxicity of IL-2 and the narrow range of tumors that respond to therapy. Cytokines that are capable of augmenting lower doses of IL-2 are, therefore, a major focus of research. We report here that granulocyte-macrophage colony-stimulating factor (GM-CSF) can augment low-dose IL-2 LAK induction from murine splenocytes. Anti-tumor necrosis factor alpha (anti-TNF alpha) or anti-interferon gamma (anti- IFN gamma) monoclonal antibodies did not inhibit (IL-2 + GM-CSF)- induced LAK generation, indicating that GM-CSF augmentation does not require TNF alpha or IFN gamma activity. Depletion of natural killer cells before culture did not inhibit low-dose IL-2-induced LAK generation or the ability of GM-CSF to augment LAK generation. In contrast, depletion of both CD4+ and CD8+ T cells before culture inhibited the generation of LAK activity. However, depletion of only CD4+ T cells, or only CD8+ T cells, did not inhibit the generation of IL-2 or (IL-2 + GM-CSF) LAK activity. These results suggest that LAK precursors are present in both the CD4+ and CD8+ T-cell populations and suggest that the addition of GM-CSF to low-dose IL-2 may result in the generation of T-derived LAK cells.

On the Relative Roles of Interleukin-2 and Interleukin-10 in the Generation of Lymphokine-Activated Killer Cell Activity

Induction of cytokine gene transcription by recombinant human IL-2 (rhIL-2) in peripheral blood mononuclear cells (PBMC) from healthy donors was studied by qualitative polymerase chain reaction. In all donors tested, optimal doses of rhIL-2-induced transcription of genes encoding for IL-5, GM-CSF, IFN-γ and TNF-α whereas transcription of IL-1-α, IL-3, IL-4, and IL-6 genes could only be detected in about half of the donors. Moreover, we observed that different doses of rhIL-2 were needed to induce transcription of different cytokine genes. In contrast, transcription of IL-2 and IL-10 genes was only observed in a minority of donors, irrespective of the concentration of rhIL-2 used. Since IL-10 displays a well-characterized inhibitory activity on the synthesis of cytokines possibly involved in the generation of lymphokine-activated killer (LAK) cells, we asked whether the absence of IL- 10 gene transcription plays a role in the induction of LAK cells. Thus, we tested the effects of different doses of rhIL-10 on the rhIL-2-driven generation of LAK activity. Interestingly, rhIL-10 dose-dependently inhibited the production of IFN-γ and TNF-α induced by IL-2, but had no effects on PBMC proliferation and generation of LAK activity. Similarly, purified CD3-/CD16+ lymphocytes, the precursors of LAK effector cells, could be optimally induced by low doses of rhIL-2 to proliferate and generate MHC-unrestricted cytotoxic activity against NK-resistant targets in the presence of rhIL-10. Altogether, our results indicate that rhIL-2 induces transcription of a preferential pattern of cytokine genes, with the IL-10 gene being infrequently transcribed. On the other hand, rhIL-10 shows diverse effects on rhIL-2-triggered PBMC activation, in that it inhibits IFN-γ and TNF-α production but does not affect PBMC proliferation or generation of LAK activity.

Induction of lymphokine-activated killer cells from nude mouse spleen cells by interleukin-4.

The induction of lymphokine-activated killer (LAK) cells from natural killer (NK) lineage cells by interleukin-4 (IL-4) was studied in vitro. Activation of nude mouse spleen cells by IL-4 generated cytotoxic cells, capable of killing NK-sensitive as well as NK-resistant tumor cells. The induction of peak lytic activity was demonstrated after 3 days of culture with IL-4. Surface marker analysis indicated that the majority of precursor cells were aGM1+, Thy1-, and the majority of effector cells were aGM1+, Thy1+, suggesting that IL-4 induced LAK cells from nude mouse spleen cells were similar to those from normal mouse spleen cells. The induction of nude mouse LAK cells by IL-4 was partially inhibited by anti-IL-4 or anti-interferon (IFN)-alpha,beta antibody, and it was further inhibited by the combination of two antibodies, suggesting that IFN-alpha,beta production was associated with LAK induction of NK lineage cells by IL-4.

Effect of ASTA-Z 7575 (INN Maphosphamide) on human lymphokine-activated killer cell induction.

Recent studies combining chemotherapeutic agents with various biological response modifiers for the treatment of cancer have shown promising results. Cyclophosphamide (Cy) is the most widely used alkylating agent and a major constituent of combination chemotherapy regimens for many neoplastic diseases. It has been reported that Cy is a cytotoxic drug, which becomes immunosuppressive at higher doses. A synthetic metabolite of Cy, ASTA-Z, has recently been produced. ASTA-Z is more active and stable by itself and does not need to be metabolically converted to an active compound. The combined effect of Cy and interleukin-2 (IL-2) on the induction of lymphokine-activated killer (LAK) cells is not known. Therefore, we decided to investigate the effect of ASTA-Z on the induction and function of LAK. The coculture of peripheral blood mononuclear cells (PBMC) with various concentrations of ASTA-Z (0, 10(-6), 10(-5), 10(-4), and 10(-3) dilution) and IL-2 (50 U/ml) for 4 days produced significant suppression of cytotoxicity and lytic ability of the LAK cells against NK-sensitive (K562) and NK-resistant (M14) tumor cell lines. The lower doses of ASTA-Z did not affect the generation of LAK cells, its cytotoxicity and lytic ability of ASTA-Z against both NK-sensitive and NK-resistant tumor cell lines. Furthermore, the ASTA-Z produced dose-dependent suppression of the proliferative response of LAK cells. The significant therapeutic benefit in the cancer patient may be achieved by the low dose regimen of Cy and IL-2 because it has no deleterious effect on the induction and function of LAK cells.

Regulation of lymphokine-activated killer cell induction by human recombinant IL-1 receptor antagonist. Obligate paracrine pathway of IL-1 during lymphokine-activated killer cell induction.

IL-2-stimulated human lymphocytes, referred to as lymphokine-activated killer (LAK) cells, can develop a broad range of lytic activity against fresh tumor cells and cultured tumor cell lines. IL-1, a pleiotropic cytokine shown to synergize with IL-2 on LAK induction, is endogenously synthesized and secreted by LAK cells. Immunoblot analysis demonstrated that IL-2-stimulated PBL produced the 31- to 34-kDa pro-molecules of IL-1 within 24 h and maintained their expression for at least 96 h. The role of secreted IL-1 has been examined using rIL-1R antagonist (IL-1ra). The addition of IL-1ra to LAK activation culture resulted in dose-dependent inhibited lytic activity, which was more apparent in LAK cells cultured with higher doses of IL-2. However, IL-1ra had no effect on proliferative responses elicited in LAK cells by IL-2. Moreover, when IL-1 binding was blocked by IL-1ra, the expression of the IL-2R p55 subunit was reduced compared with control LAK cells. The effect of IL-1 binding blockade on expression of other cytokine mRNA was further examined by polymerase chain reaction analysis, and, specifically, inhibition of both TNF-alpha and TNF-beta mRNA expression by IL-1ra was observed in PBL stimulated with IL-2. The reduced biologic activity of TNF in culture supernatants correlated well with the inhibition of mRNA expression. These findings suggest that autocrine/paracrine IL-1 is involved in the initial generation of LAK activity and, in particular, that TNF expression could be induced via an IL-1 autocrine pathway.