Effectivein vivo induction of lymphokine-activated killer (LAK) cells by pretreatment with a streptococcal preparation,OK-432

Effects of a streptococcal preparation, OK-432, on precursors of lymphokine-activated killer (LAK) cells were observedin vivo. Total number of splenocytes and the ratio of asGM ₁ ⁺ cells increased gradually after i.v. administration of OK-432, reaching their peaks at 3 to 4 days. It was found that as GM ₁ ⁺ cells were nonadherent and large in size. There were little differences in the ratios of Thy-1⁺, Lyt-2⁺, and L3T4⁺ cells before and after OK-432 treatment. Mice were injected i.p. with recombinant interleukin 2 (rIL-2) at a dose of 5 × 10⁴ U per mouse 4 days after OK-432 administration and LAK activity in their splenocytes was examined using natural killer (NK) resistant EL-4 target cells. Splenocytes in mice treated with both OK-432 and rIL-2 showed higher LAK activity than those in mice treated with rIL-2 alone.In vivo treatment with anti asGM, antibody prior to rIL-2 injection abolished completely such augmentation of LAK activity in OK-432 treated mice. These results demonstrated that asGM ₁ ⁺ LAK precursor cells induced by OK-432 were effectively differentiated into LAK cells by rIL-2.     

Antiviral effect of lymphokine-activated killer cells: characterization of effector cells mediating prophylaxis

Lymphokine-activated killer (LAK) cells generated by cultivation of C57BL/6 mouse spleen cells in the presence of recombinant interleukin-2 were transferred into natural killer (NK) cell-deficient suckling mouse recipients. These mice were then challenged with either murine cytomegalovirus (MCMV) or lymphocytic choriomeningitis (LCMV) and sacrificed 3 days later. No interleukin 2 infusions were given. Mice receiving as few as 5 x 10(5) LAK cells had several 100-fold decreases in spleen MCMV titers as compared with untreated mice. This treatment had no effect on spleen LCMV titers. The LAK cell cultures contained 10 to 17% NK 1.1+, 50 to 55% Lyt-2+, and 33 to 50% immunoglobulin D+ cells. Double fluorescence labeling and in vitro cytotoxicity assays with fluorescence-activated cell sorting revealed at least two mutually exclusive killer cell populations. NK 1.1+ LAK cells resembled freshly isolated activated NK cells with regard to target cell range (YAC-1 cell killing greater than L-929, P815, and EL-4 cell killing), large granular lymphocyte (LGL) morphology, and decreased ability to lyse interferon (IFN)-treated target cells. Lyt-2+ LAK cells lysed the targets mentioned above but at lower levels and without the differences in susceptibility mentioned above. These Lyt-2+ LAK cells also had a decreased ability to lyse IFN-treated targets, in contrast to classic cytotoxic T lymphocytes, which lyse IFN-treated targets far more efficiently than untreated targets. Purified populations of LAK cells obtained by fluorescence-activated cell sorting were used in the antiviral protection model. The results showed that protection against MCMV could be mediated by NK 1.1+, NK 1.1-, Lyt-2+, Lyt-2-, and IgD- populations but not by IgD+ cells. The five protective populations all had in common the LGL phenotype and cytotoxic activity in vitro. The IgD+ population did not contain LGLs, lyse target cells in vitro, or mediate an antiviral effect in vivo. These results suggest that LAK cells may be therapeutically useful against certain virus infections (MCMV) but not others (LCMV) and that despite their heterogeneity in antigenic phenotype and cytotoxic activity, their pattern of antiviral activity in vivo resembles that of NK cells, which protect against MCMV but not LCMV.     

In vivo effects of recombinant IL-2. I. Isolation of circulating Leu-19+ lymphokine-activated killer effector cells from cancer patients receiving recombinant IL-2

This study was designed to isolate and phenotypically characterize lymphokine-activated killer (LAK) cells generated in vivo during administration of high dose rIL-2 to cancer patients. The development of circulating LAK effector cells in these patients was demonstrated by the ability of fresh PBL to exhibit lytic activity against the NK-resistant Daudi cell line and fresh tumor cells without prior in vitro culture with rIL-2. Kinetic studies demonstrated that circulating LAK effector cells are detectable 4 to 6 wk after the initiation of rIL-2 therapy. Cells isolated by FACS revealed that circulating LAK cells are Leu-19+, Leu-17+ but CD5-. We have previously reported that circulating Leu-19+ cells are heterogeneous with regard to the expression of CD16 and CD8. Since sorting of cells expressing Leu-19 and either low quantities of CD8 or CD16 resulted in cytolytic activity in both the positive and negative fractions, these latter two markers do not identify subpopulations of Leu-19+ cells with or without LAK cytolytic activity. Although all LAK cells generated in vivo were Leu-19+, we generated LAK cells from the Leu-19- subpopulation after in vitro culture with rIL-2, suggesting that at least some of in vitro generated LAK cells are derived from Leu-19- precursor cells. These LAK cells did not, however, express the Leu-19 surface marker. Based on the functional data reported in this paper, we conclude that circulating LAK effector cells are a phenotypically heterogeneous population that express surface Ag in association with NK cells and not T lymphocytes.     

Augmentation by anti-T3 antibody of the lymphokine-activated killer cell-mediated cytotoxicity

This study showed that a mAb (145-2C11) against the T3 epsilon-chain of the TCR complex augmented the cytotoxic activity of the lymphokine-activated killer (LAK) effectors. The LAK cells were induced by culturing normal spleen cells with purified human rIL-2. Adding alpha T3 at the effector phase of the cytotoxic reactions augmented the LAK-mediated cytotoxicity. The alpha T3-augmented LAK killing was seen only with tumor targets, and there was no increase of killing against Con A-induced lymphoblasts. The augmentation effect was dose dependent on both the amounts of alpha T3 and the number of LAK cells added. A very low concentration of alpha T3 (1/10,000 dilution of culture supernatants) was sufficient to induce alpha T3-augmented LAK-mediated cytotoxicity. Human rIL-2 at 10 to 30 U/ml was sufficient to generate LAK cells for maximal alpha T3 augmentation, whereas 300 to 1000 U/ml of IL-2 were needed to generate maximal LAK activity when tested in the absence of alpha T3. LAK cells generated for longer periods of time showed a progressive increase of alpha T3-augmented cytotoxicity. For some targets, the alpha T3-augmented LAK killing was FcR dependent as evidenced by the ability of alpha FcR mAb 2.4G2 to inhibit, and for others it was not inhibited. The alpha T3-augmented killing did not correlate with the FcR expression on target cells as defined by 2.4G2. The LAK cells were both Lyt-2+ and Lyt-2-, but the LAK cells involved in alpha T3-augmented killing were exclusively Lyt-2+. Preincubation of LAK cells with alpha T3, but not preincubation of targets with alpha T3, resulted in augmented killing suggesting that the alpha T3 effect was unrelated to an antibody-dependent cell-mediated cytotoxicity. Our findings indicate that alpha T3 is a potent reagent to augment the cytotoxic reaction of LAK cells. These results suggested that a relationship might exist between the T3 complex and the cytotoxic activity of a subpopulation of Lyt-2+ LAK cells.     

Generation of lymphokine-activated killer (LAK) cells from tumor-infiltrating lymphocytes

Culture of tumor-infiltrating lymphocytes (TIL) containing about 20% BMC2 tumor cells with recombinant human interleukin 2 (rIL-2) resulted in the diminish of tumor cells and the growth of lymphocytes. These IL-2-activated lymphocytes showed a strong cytotoxic activity against not only syngeneic tumor cells but also allogeneic tumor cells. Such broad-reactive killer cells, termed lymphokine-activated killer (LAK) cells, are also inducible from spleen cells by in vitro activation with IL-2. However, LAK cells generated from TIL (TIL-LAK) showed higher cytotoxic activity against BMC2 than LAK cells generated from spleen cells (S-LAK). Furthermore, it was demonstrated that TIL-LAK cells revealed marginal cytotoxic activity against normal Con A blasts and YAC-1 cells as opposed to S-LAK. Flow cytometric analysis of TIL-LAK indicated that TIL-LAK cells mainly consisted of Thy 1.2+, Ly 2+, asialo GM1+ cells. TIL-LAK cells displayed not only in vitro cytotoxicity but also in vivo anti-tumor activity. Furthermore, it was also confirmed that TIL-LAK cells could be induced in autochthonous mouse tumor systems and human gastric tumor systems.     

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.     

IL-4 inhibits IL-2-mediated induction of human lymphokine-activated killer cells, but not the generation of antigen-specific cytotoxic T lymphocytes in mixed leukocyte cultures

Human rIL-4 was studied for its capacity to induce lymphokine-activated killer (LAK) cell activity. In contrast to IL-2, IL-4 was not able to induce LAK cell activity in cell cultures derived from peripheral blood. IL-4 added simultaneously with IL-2 to such cultures suppressed IL-2-induced LAK cell activity measured against Daudi and the melanoma cell line MEWO in a dose-dependent way. IL-4 also inhibited the induction of LAK cell activity in CD2+, CD3-, CD4-, CD8- cells, suggesting that IL-4 acts directly on LAK precursor cells. IL-4 added 24 h after the addition of IL-2 failed to inhibit the generation of LAK cell activity. Cytotoxic activity of various types of NK cell clones was not affected after incubation in IL-4 for 3 days, indicating that IL-4 does not affect the activity of already committed killer cells. No significant differences were observed in the percentages of Tac+, NKH-1+ and CD16+ cells after culturing PBL in IL-2, IL-4 or combinations of IL-2 and IL-4 for 3 days. IL-4 also inhibited the activation of non-specific cytotoxic activity in MLC, as measured against K-562 and MEWO cells. In contrast, the Ag-specific CTL activity against the stimulator cells was augmented by IL-4. Collectively, these data indicate that IL-4 prevents the activation of LAK cell precursors by IL-2, but does not inhibit the generation of Ag-specific CTL.     

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.     

Generation of MC-38 adenocarcinoma tumor-specific tumor infiltrating lymphocytes by murine anti-CD3 antibody and recombinant interleukin-2.

The growth, in vitro cytolytic activity and phenotype of murine MC-38 adenocarcinoma tumor infiltrating lymphocytes (TILs) stimulated with anti-CD3 monoclonal antibody (mAb) and recombinant interleukin-2 (RIL-2) as compared to RIL-2 alone was investigated. When assayed for growth, anti-CD3 mAb + RIL-2 MC-38 TILs demonstrated an enhanced proliferative activity compared to RIL-2 alone (fold expansion, 16,228 and 365,713 compared to 112 and 5594, culture times: 55 and 118 days, experiments 1 and 2, respectively). TILs cultured with anti-CD3 mAb alone demonstrated little expansion (fold expansion 6 and 3, experiments 1 and 2, respectively). Early during culture, the anti-CD3 mAb + RIL-2 MC-38 expanded TILs demonstrated broad cytolytic activity (LU: day 17, against MCA-102: greater than 125, YAC-1: greater than 125, MC-38, greater than 125). This lytic picture reversed with time with increasing specificity demonstrated against MC-38 (LU: day 53, MCA-102: less than 1, YAC-1: less than 1, MC-38: 8). TILs expanded with RIL-2 alone demonstrated more lysis of the YAC-1 target and little lysis of the other targets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth of MCF-7 human breast carcinoma in severe combined immunodeficient mice: Growth suppression by recombinant interleukin-2 treatment and role of lymphokine-activated killer cells

Summary The severe combined immunodeficient (SCID) mouse, lacking functional T and B lymphocytes, has been considered by many groups to be a prime candidate for the reconstitution of a human immune system in a laboratory animal. In addition, this immuno-deficient animal would appear to have excellent potential as a host for transplanted human cancers, thus providing an exceptional opportunity for the study of interactions between the human immune system and human cancer in a laboratory animal. However, because this animal model is very recent, few studies have been reported documenting the capability of these mice to accept human cancers, and whether or not the residual immune cells in these mice (e.g. natural killer, NK, cells; macrophages) possess antitumor activities toward human cancers. Thus, the purpose of this study was (a) to determine whether or not a human breast carcinoma cell line (MCF-7) can be successfully transplanted to SCID mice, (b) to determine whether or not chronic treatment of SCID mice with a potent lymphokine (recombinant interleukin-2, rIL-2) could alter MCF-7 carcinoma growth, and (c) to assess whether or not rIL-2-activated NK cells (LAK cells) are important modulators of growth of MCF-7 cells in SCID mice. To fulfill these objectives, female SCID mice were implanted s.c. with MCF-7 cells (5 × 10⁶ cells/mouse) at 6 weeks of age. Six weeks later, some of the mice were injected i.p. twice weekly with rIL-2 (1 × 10⁴ U mouse^–1 injection^–1). Results clearly show that MCF-7 cells can grow progressively in SCID mice; 100% of the SCID mice implanted with MCF-7 cells developed palpable measurable tumors within 5–6 weeks after tumor cell inoculation. In addition, MCF-7 tumor growth was significantly (P <0.01) suppressed by rIL-2 treatment. rIL-2 treatment was non-toxic and no effect of treatment on body weight gains was observed. For non-tumor-bearing SCID mice, splenocytes treated in vitro with rIL-2 (lymphokine-activated killer, LAK, cells) or splenocytes derived from rIL-2-treated SCID mice (LAK cells) had significant (P <0.01) cytolytic activity toward MCF-7 carcinoma cells in vitro. In contrast, splenocytes (LAK cells) derived from tumor(MCF-7)-bearing rIL-2-treated SCID mice lacked cytolytic activities toward MCF-7 cells in vitro. No significant concentration of LAK cells in MCF-7 human breast carcinomas was observed nor did rIL-2 treatment significantly alter growth of MCF-7 cells in vitro. Thus, while rIL-2 treatment significantly suppressed growth of MCF-7 breast carcinomas in SCID mice, the mechanism of this growth suppression, albeit clearly not involving T and B lymphocytes, does not appear to be mediated via a direct cytolytic activity of LAK cells toward the carcinoma cells. However, rIL-2-activated SCID mouse splenocytes (LAK cells) do possess the capability of significant cytolytic activity toward MCF-7 human breast carcinoma cells. Thus, treatment of SCID mice with a potent lymphokine (rIL-2) induces a significant antitumor host response, a response that does not involve T and B lymphocytes and appears not to involve NK/LAK cells. This host response must be considered in future studies designed to investigate the interactions of reconstituted human immune systems and human cancers within this highly promising immuno deficient experimental animal model.     

Analysis of the murine lymphokine-activated killer (LAK) cell phenomenon: dissection of effectors and progenitors into NK- and T-like cells

Murine as well as human lymphokine-activated killer (LAK) cells have been reported to have several characteristics of T lymphocytes and to be clearly distinct from natural killer (NK) cells. The present study of murine LAK cells showed that cytotoxic cells generated in the presence of interleukin 2 IL 2 were heterogeneous with respect to cell surface markers of progenitor as well as effector cells. Negative selection of cells with antibodies and complement or positive selection by fluorescence-activated cell sorting unequivocally showed that LAK effector cells consisted of at least two clearly distinct populations, the relative contribution of which was dependent on donor organ and target cells studied. Approximately 40% of the cytotoxic activity of spleen-derived effector cells active against the NK-resistant targets EL-4 or MCA-5 was eliminated by treatment with antibodies to the NK-markers asialo-GM1 and NK 1 (NK-LAK). Approximately 60% of cytotoxic activity was associated with cells expressing the T cell marker Lyt-2, lacked NK 1, and was lacking or expressed only small amounts asialo-GM1 (T-LAK). The NK-LAK cells were of greater importance for the cytotoxic activity against the standard NK target YAC-1, although T-LAK cells also excerted significant cytotoxicity against this cell line. Limiting dilution analysis estimated that the minimal frequency of precursors developing into cells with cytotoxic activity against EL-4 was 1/6700 in spleen and 1/4200 in peripheral blood. The frequency of cells developing into cytotoxic effectors against YAC-1 cells was 1/3700 and 1/1450 in spleen and peripheral blood, respectively. Depletion of progenitor cells from spleen or peripheral blood expressing NK 1 or Lyt-2 by treating the cells with antibodies to these structures and complement indicated that NK-1-expressing cells were the dominating progenitor of the LAK cells irrespective of target cells used. Culture of murine lymphoid cells from spleen or peripheral blood with high concentrations of IL 2 results in the emergence of two different killer cell populations with phenotypic similarities to NK and T cells, respectively, both being able to kill targets resistant to resting NK cells. In contrast to numerous earlier reports, we concluded that LAK cells are heterogeneous with respect to surface markers, with a major population of LAK cells apparently representing IL 2-activated cells expressing cell surface markers associated with NK cells.     

Immunotherapy of intraperitoneal cancer with interleukin 2 and lymphokine-activated killer cells reduces tumor load and prolongs survival in murine models

The control of malignancy disseminated within the peritoneal cavity is an important problem in the management of low-grade gastrointestinal and ovarian neoplasms. A model of peritoneal carcinomatosis in the mouse was used to investigate the potential of lymphokine-activated killer (LAK) cells and exogenous interleukin 2 (IL-2) to control intraperitoneal tumor. LAK cells are splenocytes activated in vitro by IL-2. C57BL/6 mice were injected intraperitoneally with a lethal inoculum of syngeneic MCA-105 tumor. Three days later, the established tumor was treated with adoptively transferred LAK cells and/or exogenous IL-2 administration. LAK cells alone were ineffective in reducing intraperitoneal tumor. Administration of IL-2 alone resulted in limited tumor reduction. Treatment with exogenous IL-2 in conjunction with LAK cells resulted in the greatest reduction of intraperitoneal tumor. The larger the number of LAK cells given, the greater the reduction in tumor. Frequent intraperitoneal bolus administration of IL-2 was more effective than a single daily intraperitoneal injection and intraperitoneal administration of IL-2 and LAK was more effective than systemic treatments. Marked prolongation of life was seen in mice treated with LAK cells plus exogenous IL-2. We conclude that intraperitoneal LAK cells plus exogenous IL-2 is an effective treatment regimen for reducing intraperitoneal tumor in this murine model.     

The anti-tumor efficacy of lymphokine-activated killer cells and recombinant interleukin 2 in vivo: direct correlation between reduction of established metastases and cytolytic activity of lymphokine-activated killer cells

Our previous studies demonstrated that the incubation of human peripheral blood lymphocytes or murine splenocytes in recombinant interleukin 2 (RIL 2) resulted in the generation of lymphokine-activated killer (LAK) cells capable of lysing a broad spectrum of fresh tumors in short-term chromium-release assays. Moreover, injections of LAK cells plus RIL 2 were highly effective in eliminating established 3 day metastases in the lung and liver (1-3). We have examined several parameters to define whether or not the cytolytic activity of LAK cells as measured in vitro correlated directly with the in vivo anti-tumor efficacy of adoptively transferred LAK cells. LAK cells plus RIL 2 could mediate marked reductions of established pulmonary metastases in mice rendered T cell deficient by adult thymectomy and lethal, total body irradiation followed by reconstitution with T cell-depleted bone marrow and spleen cells. Thus there was no requirement for additional T lymphocytes of host origin for successful therapy with adoptively transferred LAK cells plus RIL 2. Fresh splenocytes depleted of T cells by anti-Thy-1.2 monoclonal antibody plus complement generated LAK cells that were as highly lytic to fresh tumor in vitro and were as effective in reducing established pulmonary metastases as those generated from untreated or complement-treated splenocytes. Thus the precursor to LAK cells with anti-tumor activity in vitro and in vivo did not express the Thy-1 antigenic marker. In contrast, treatment of LAK effector cells (those generated from a 3-day incubation of fresh, normal splenocytes in RIL 2) with anti-Thy-1.2 antibody plus complement reduced or abolished their in vitro cytolytic activity. However, when combined with the systemic administration of RIL 2, these T cell-depleted, non-lytic LAK cells remained as effective in reducing the number of established pulmonary metastases upon adoptive transfer as untreated or complement-treated lytic LAK cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transforming growth factor-beta inhibits the in vitro generation of lymphokine-activated killer cells and cytotoxic T cells

Summary The effect(s) of purified transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) on the induction and function of lymphokine-activated killer (LAK) cells and cytotoxic T lymphocytes (CTL) was examined. The addition of TGF-beta, but not PDGF, to cultures containing fresh C57BL/6 mouse splenocytes or human peripheral blood lymphocytes plus recombinant interleukin-2 markedly inhibited the development of mouse and human LAK cell activity (measured after 3 days for cytotoxicity against cultured or fresh tumor targets in 4-h ⁵¹Cr release assays). The addition of TGF-beta, but not PDGF, to a one-way, C57BL/6 anti-DBA/2, mixed lymphocyte reaction effectively blocked the generation of allospecific CTL as well. However, TGF-beta did not inhibit the effector function of LAK cells or of allospecific CTL when added directly to the short-term cytolytic assay. A second form of homodimeric TGF-beta, type 2, was also found to be suppressive on the development of murine LAK cells and allospecific CTL. Collectively, these data demonstrate that the peptide TGF-beta is a potent inhibitor of LAK cell and CTL generation in vitro.     

Heterogeneous lymphokine-activated killer cell precursor populations

Summary We developed a monoclonal antibody (mAb) 211, which recognizes the precursors in peripheral blood of lymphokine-activated killer cells (LAK) induced by recombinant interleukin-2 (rIL-2). In conjunction with complement mAb 211 also eliminates natural killer cells (NK) and a majority of the cytotoxic T lymphocytes. B cells and monocytes do not express the 211 antigen. Since mAb 211 recognized such a large percentage of peripheral blood lymphocytes we examined which 211⁺ subpopulation was the predominant precursor of rIL-2-induced LAK cells using two-color fluoresence-activated cell sorting (fluorescein-conjugated 211 mAb plus phycoerythrin-CD11b). This method identified the 211⁺/ CD11b⁺ population as the predominant phenotype of the rIL-2-induced LAK precursor. In addition, we directly compared the phenotype of the LAK precursor induced by delectinated T-cell growth factor (TCGF) to that induced by rIL-2. The 211-depleted population, which was devoid of NK cells and LAK precursors (inducible by rIL-2), was capable of generating LAK activity when TCGF was used as the source of lymphokine. LAK cells induced by TCGF from the 211-depleted population lysed a fresh sarcoma and an NK-resistant cultured melanoma tumor target but not the Daudi cell line, which was lysed by rIL-2-induced LAK cells. Lymphoid subpopulations, depleted using NKH1a mAb, behaved similarly, generating high levels of lysis against the two solid tumor targets when cultured with TCGF but not with rIL-2. CD 3-depleted populations showed enrichment for LAK precursors using either rIL-2 or TCGF. These results indicate that while rIL-2-induced LAK precursors cannot be separated from cells with NK activity, TCGF-induced LAK cells can be generated from populations of peripheral blood mononuclear cells without NK activity.     

IL-4-induced lymphokine-activated killer cells. Lytic activity is mediated by phenotypically distinct natural killer-like and T cell-like large granular lymphocytes

The purpose of the current study was to characterize lymphokine-activated killer (LAK) activity induced with IL-4/B cell stimulatory factor-1 and to compare IL-4-induced LAK activity with IL-2-induced LAK activity. Culture of murine lymphocytes with high concentrations of IL-4 induced nonspecific lytic activity against a wide variety of tumors. Lytic activity induced by IL-4 increased with increasing concentrations of IL-4 over the range of 1.0 to 25 ng/ml. The kinetics of LAK induction by IL-4 and IL-2 were similar; however, IL-4 was less effective than IL-2 in maintaining lytic activity for longer culture periods and provided lower viable cell yields than did IL-2. Similar to IL-2, IL-4 induced blastogenesis and the generation of large granular lymphocytes, all LAK activity observed was exclusively associated with the large granular lymphocyte fraction, and the cytolytic effector cells were heterogeneous in regards to cell surface phenotype. The majority of IL-4-induced lytic activity was associated with mutually exclusive NK-like (i.e., NK-1.1+ Lyt-2-) and T cell-like (i.e., NK-1.1- Lyt-2+) LAK cells. The precursors for each subset were distinct and expressed the asialo-GM1+ Lyt-2- and the asialo-GM1+ Lyt-2+ phenotypes, respectively. Although IL-4-induced LAK effector cells were morphologically and phenotypically similar to IL-2-induced LAK cells, IL-2 generated equivalent numbers of T cell-like and NK-like LAK cells, whereas IL-4 generated 3.5-fold more T cell-like LAK cells than NK-like LAK cells. It might eventually be possible to exploit the preferential activation of T cell-like LAK by IL-4 for therapeutic advantage.     

Generation of lymphokine-activated killer cell activity from human thymocytes

We have generated lymphokine-activated killer (LAK) cells from human thymocytes in order to assess the relationship between LAK cells and T cells. Fresh thymocytes lack natural cytotoxic activity, and cytotoxicity cannot be stimulated by short term (1 hr) incubation with interferon or recombinant interleukin 2 (rIL-2). In addition, thymocytes are phenotypically devoid of cells bearing the natural killer (NK)-associated markers cluster designation (CD) 16 and NKH-1. After culture for 5 to 8 days with rIL-2, thymocytes display high levels of cytotoxic activity against both NK-sensitive and NK-resistant targets. Thymocytes require slightly more IL-2 than do peripheral blood lymphocytes to generate LAK activity. We have examined the phenotype of the thymocyte LAK precursor and effector cells. Thymocyte LAK precursors are of low to medium density, CD1-negative, and predominantly CD3-negative. Although CD3-positive cells proliferate in response to rIL-2, they are low in cytolytic capabilities. The effector cells, like the LAK precursors, are low to medium density lymphocytes. The cytotoxic cells are predominantly CD3-negative, and cytotoxic activity cannot be blocked with the use of anti-CD3 monoclonal antibodies. The effector cells also lack most NK-associated markers (HNK-1, and the CD16 markers Leu-11b and B73.1) but possess the NK-associated marker NKH-1 (N901). The responsive cell appears to be at a very early stage of thymic development, and it does not appear to either require or express the CD3-T cell receptor complex.     

Generation and characterization of purified adherent lymphokine-activated killer cells in mice

During the incubation of murine spleen, lymph node, or bone marrow cells with IL-2 (1000 U/ml) a small percentage of cells became adherent to the surface of plastic tissue culture flasks. After removal of the non-adherent lymphoid cells, plastic adherent lymphokine-activated killer (LAK) cells could be efficiently expanded in the presence of IL-2. Plastic adherent-derived A-LAK cells were characterized by high rates of proliferation and their cytotoxic activity was more than 10 fold higher than LAK cells generated in the bulk (unfractionated) spleen cell cultures. A-LAK cells could be continuously generated from the non-adherent cell population. Using multiple transfers (every 1 to 2 days) of non-adherent LAK cells into new flasks, new rounds of plastic adherent cells were generated with high expansion capability and high levels of cytotoxic activity. Morphologically, A-LAK cells were large granular lymphocyte and phenotypically expressed markers characteristic of NK cells (asialo GM1+, NK1.1+, Qa5+, Ly-6.2+, Thy-1.2+, but negative for Lyt-2.2 and L3T4). A-LAK cells generated from mice of different strains expressing low and high levels of NK cell activity were equally highly cytotoxic. However, A-LAK cells obtained from nude or beige mice had relatively lower levels of cytotoxicity. Stimulation of NK cell activity by poly I:C or inhibition by in vivo or in vitro treatment with anti-asialo GM1 serum did not affect the generation of A-LAK cells. A-LAK cells derived from spleen or bone marrow of C57BL/6 or nude mice treated with anti-asialo GM1 serum were found to be asialo GM1+ suggesting that A-LAK cell could be generated from the asialo GM1- precursor cells. Expansion of plastic adherent A-LAK cells in the presence of IL-2 could provide large numbers of highly purified cytotoxic A-LAK cells suitable for cancer immunotherapy.     

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.     

Precursor phenotype of lymphokine-activated killer cells in the mouse

Lymphokine-activated killer (LAK) activity has been proposed to functionally differ from natural killer (NK) activity largely on the basis of a broader target cell spectrum and different kinetics of response to interleukin 2 (IL 2). Similarly, it has been proposed that the precursor cells for LAK activity are phenotypically distinct from NK cells. In most precursor studies, phenotype comparisons have been made between fresh NK cells and LAK cells which have been generated by 3 to 5 days of culture in IL 2. In the present study, we utilized positive selection with monoclonal antibodies to characterize the surface phenotype of precursor cells which give rise to rIL 2-augmented NK activity within 24 hr and to classically generated LAK activity which appears after 3 to 5 days of culture in rIL 2. The results demonstrated that highly purified (93 to 95%) Lyt-2+ or L3T4+ T lymphocytes were unable to generate appreciable amounts of either augmented NK activity or LAK activity when cultured with rIL 2, whereas the highly purified (98%) Lyt-2-, L3T4-, asialo GM1+ lymphocyte subset gave rise to both augmented NK and LAK activities. These findings demonstrate that both augmented NK and LAK activities can arise from precursors expressing the same phenotype. Overall, the results suggest that NK cells in mouse spleen constitute a major precursor component for the generation of LAK activity from that organ.