Human lymphokine-activated killer (LAK) cells

Summary Pretreatment of peripheral blood mononuclear cells (PBMC) with 5 mMl-phenylalanine methyl ester (PheOMe) provides an efficient means to deplete monocytes. PheOMe does not affect the number of large granular lymphocytes after the pretreatment, but does inhibit natural killer cell cytotoxicity temporarily after the pretreatment. However, depletion of monocytes by PheOMe allows lymphokine-activated killer (LAK) cell generation with recombinant interleukin-2 (rIL-2) at high cell density (> 5 × 10⁶ cells/ml). The time of the PheOMe pretreatment is 40–60 min, though some effect could be observed within 15 min, and the pretreatment could be performed at room temperature. Pretreatment density of PBMC with 5 mM PheOMe could be achieved at cell density up to 3 × 10⁷ cells/ml. PheOMe-pretreated cells could be activated by rIL-2 in serumless media at high cell density. Pretreatment of PBMC with 5 mM PheOMe provides an efficient means to deplete monocytes, as compared to plastic and nylonwool adherence. LAK cell generation is similar in both methods of monocyte depletion; therefore, depletion of monocytes allows, LAK cell generation at high cell density. The PheOMe procedure provides an improved and convenient process for preparing LAK cells for adoptive immunotherapy.     

Murine lymphokine-activated killer (LAK) cells: phenotypic characterization of the precursor and effector cells

Murine and human lymphocytes incubated in recombinant interleukin 2 (RIL 2) generate a population of cytotoxic cells (lymphokine-activated killer cells [LAK]), which are able to lyse a wide array of fresh tumor cells but do not lyse fresh normal cells. Intravenous administration of these cells with the concomitant administration of RIL 2 can eliminate established pulmonary and hepatic metastases in mice. To characterize the cell that has in vitro LAK activity, we subdivided murine lymphocytes by lysing select subpopulations with the use of complement and antibodies against lymphocyte surface markers or by fluorescence-activated cell sorting. Thy-1.2-negative splenocytes were found to generate near normal amounts of LAK activity after RIL 2 incubation. Small and inconsistent LAK cell activity was generated from Thy-1.2-positive splenocytes. Ia-positive and surface immunoglobulin-positive splenocytes had little or no LAK precursor capability and did not appear to be necessary for LAK activation. Treatment of splenocytes with anti-asialo GM1 (anti-ASGM1) heterosera and complement markedly decreased their ability to generate LAK activity. At the effector stage, cytotoxic cells were of the Thy-1.2-positive, Ia-negative phenotype. Ia-depleted cells were separated into subpopulations bearing or not bearing the gamma Fc receptor (gamma FcR). The majority of cytotoxicity resided in gamma FcR-positive cells. Thus the precursors of murine LAK cells are "null" lymphocytes bearing neither T nor B cell surface markers but develop the Thy-1.2 cell surface marker in vitro, in association with the development of lytic activity for fresh tumor cells after stimulation by RIL 2.     

Human lymphokine-activated killer cells (LAK cells) as a potential immunotherapeutic modality

Lymphokine-activated killer cells (LAK) are cytolytic lymphocytes with the unique capacity of killing NK-resistant fresh human tumor cells in short-term assays. LAK appear to kill autologous tumors as well as TNP-modified self and allogeneic tumors with complete crossreactivity, both at the population and clonal level. Initial studies on the classification of LAK conclude that LAK are distinct from the classical NK and T-lymphocyte systems based on a number of criteria including surface phenotype, activation conditions, and spectrum of susceptible target cells. LAK kill rasoncogene-transfected fibroblasts in a manner similar to fresh tumors. As yet, the target cell determinant responsible for susceptibility to LAK lysis is unknown, but cell-surface proteins are definitely involved. Activation of LAK requires only IL-2, and is most efficient using serum-free conditions. Because interleukin-2 alone is sufficient for LAK activation, we have tested in vitro whether fresh PBL could be activated in the presence of tumor, as might be desired in vivo. LAK activation was greatly suppressed by tumor presence. LAK activation is also suppressed by hydrocortisone, but not cyclosporine A. Because of the above and other findings, we have initiated a clinical protocol to test whether LAK made from brain-tumor patients' PBL could eliminate residual glioma tumor cells. Autochthonous LAK, plus rIL-2 to maintain lytic ability, are injected during surgery. Preclinical studies in a rat glioma model have shown this approach to be safe. Eleven glioma patients have been injected intracerebrally with IL-2 and/or LAK with no immediate or long-term (14 months follow-up) adverse effects. Much work is needed to understand the LAK phenomenon and to resolve its potential usefulness in cancer therapy as well as its inherent biologic role.     

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.     

Human oncogene-transfected tumor cells display differential susceptibility to lysis by lymphokine-activated killer cells (LAK) and natural killer cells

NIH 3T3 tertiary transfectants containing the N-ras or c-Ha-ras oncogenes derived from human tumors were tested for susceptibility to lymphokine-activated killer (LAK) cell and natural killer (NK) cell lysis. N-ras tertiary transfectants contained a human acute lymphocytic leukemia-derived N-ras oncogene. C-Ha-ras transfectants contained either the position 61-activated form of the oncogene (45.342, 45.322, and 45.3B2) or the position 12-activated form (144-162). In 4 hr 51Cr release assays, seven of seven in vivo grown human oncogene transfected NIH 3T3 fibroblasts were lysed by murine LAK effectors, whereas six of seven were lysed by human LAK effectors. There was no difference in susceptibility to lysis between cells transfected with the N-ras oncogene, the position 61 activated c-Ha-ras oncogene, or the position 12 activated c-Ha-ras oncogene. Cultured NIH 3T3 fibroblasts, as well as in vitro and in vivo grown NIH 3T3 tertiary transfectants were resistant to lysis by murine NK effectors and were relatively resistant (4/6 were not lysed) to lysis by human NK effectors. We conclude that human oncogene-transfected tumors are susceptible to lysis by both murine and human LAK cells while being relatively resistant to lysis by murine and human NK cells. Different oncogenes or the same oncogene activated by different point mutations do not specifically determine susceptibility to lysis by LAK or NK. Also the presence of an activated oncogene does not appear to be sufficient for inducing susceptibility to these cytotoxic lymphocyte populations.     

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

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

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

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

Interferon-gamma-treated K562 target cells distinguish functional NK cells from lymphokine-activated killer (LAK) cells

In vitro incubation of the erythroleukemic cell line K562 with interferon-gamma (IFN-gamma) renders these cells relatively resistant to natural killer (NK) cell lysis. However, such treatment does not alter their sensitivity to LAK cell lysis. Thus, the lytic susceptibility of interferon-gamma-treated K562 (I-K562) cells to LAK cells as opposed to its relative resistance to NK cell lysis provides a functional assay to help distinguish these two types of effector cells. The relative resistance of I-K562 for NK cell-mediated lysis was not secondary to the release of soluble factors or the frequency of Leu-19+, CD3+ T cells, residual IFN-gamma, or expression of MHC Class I molecules. Coincubation of I-K562 cells with NK or LAK cells overnight did not appreciably change the pattern of lytic responses against K562 and I-K562 target cells. However, incubation of PBMC in vitro with I-K562 but not native K562 in the presence of r-IL-2 leads to a marked decrease in the generation of LAK cells. The inhibition of LAK cell generation was not secondary to differences in the consumption of bioactive levels of IL-2. Differences in the lytic capability of NK and LAK effector cells suggest heterogeneity among cells that mediate such non-MHC-restricted lysis. Use was made of cells from a patient with a large granular lymphocyte lymphoproliferative disease (greater than 85% Leu-19+) to determine if such cells could be used to distinguish clonal population of cells which would represent NK or LAK cell function. Of interest was the finding that such cells, even after incubation in vitro with IL-2, showed lytic function representative of NK cells but not LAK cells. Data concerning the inhibition of LAK cell generation by I-K562 cells have important implications for future therapeutic trials of IFN-gamma and IL-2 in the treatment of human malignancies.     

小鼠LAK细胞对粒—单系祖细胞增殖的影响

Murine lymphokine-activated killer (LAK) cells were generated from spleen cells of C57/BL6 mice by culture of spleen cells in vitro for 72 hours in medium containing 500 units/ml recombinant human interleukin 2 (IL-2), and effects of these LAK cells on proliferation of syngenic myeloid progenitor cells (CFU-GM) were observed. After 3 days culture, LAK cells were assayed for their cytotoxicity in a 4 hours 51Cr-release test. Either natural killer (NK) cell sensitive YAC-1 lymphoma cells or NK cell resistant LP-3 and WEHI-164 fibrosarcoma cells were efficiently lysed by murine LAK cells. When LAK cells were added into culture system in a final concentration of 5 x 10(4)/ml, 2 x 10(5)/ml, 8 x 10(5)/ml, CFU-GM were increased by 55.2%, 165.5%, and 194.4% of control respectively. LAK-CM also showed augmentative effect on CFU-GM growth. When 10% (v/v) of LAK-CM were added into culture system, CFU-GM were increased by 51.4% of control, but LAK-CM alone could not stimulate CFU-GM growth. Again, effects of LAK-BMC interaction on CFU-GM formation were investigated. CFU-GM were inhibited to 27.6% of control when 1 x 10(5) BMC were mixed with 8 x 10(5) LAK cells and incubated for 4 hours prior to CFU-GM culture. These data suggest that (1) LAK cells may secrete co-CSF which showed synergistic effect with CSF on CFU-GM proliferation: (2) When LAK cells contact with BMC, they showed significant cytotoxicity to myeloid progenitor cells which mediated decrease of CFU-GM formation.     

Human lymphokine-activated killer cells: further isolation and characterization of the precursor and effector cell

In a series of experiments we have demonstrated the progressive enrichment (5- to 40-fold) in lymphokine-activated killer (LAK) precursor activity by adherence depletion, sheep red cell rosetting, and depletion of CD3- and DR-positive lymphocytes. The LAK precursor cell thus appears to fall within the 'null' cell population. CD16 and CD11 are cell surface antigens expressed on the surface of the LAK precursor as demonstrated in sorting experiments. A 6- to 100-fold enrichment compared to unseparated peripheral blood was noted when sorted cells positive for CD16 and CD11 were tested. The LAK effector has been identified as being primarily CD3- and CD2+. Similar sorting equipment demonstrated a 7- to 500-fold difference in lytic activity for fresh tumor when comparing CD2+/CD3- and CD2+/CD3+ cells. The CD16+/CD11+ lymphocyte can proliferate in response to interleukin-2 (IL-2) alone in the absence of accessory cells and can be expanded in IL-2 alone with maintenance of lytic activity.     

Phenotypic and functional analysis of lymphokine-activated killer (LAK) cell clones

Summary Lymphokine-activated killer (LAK) cells are generated by the culture of peripheral blood lymphocytes with interleukin-2 (IL-2). A variety of cells, including T-lymphocytes and natural killer (NK) cells, can be activated by IL-2 to exhibit the ability to kill multiple tumor and modified-self targets. Recent reports indicate that culture conditions can determine the phenotype of cells expressing LAK activity. Using limiting dilution techniques, we first generated cloned LAK cells with three culture conditions: autologous human serum (AHS)+IL-2; AHS+IL-2+0.1 g/ml phytohemagglutinin and fetal bovine serum and IL-2. We determined that all but one of the 47 LAK cell clones generated with the three culture conditions were CD3⁺ and T-cell like; one NK-like clone was observed. Clones that were cytotoxic for one target could generally kill multiple targets, and the absence of phytohemagglutinin did not significantly affect the ability of the LAK cell clones to kill multiple targets. The presence of phytohemagglutinin was, however, necessary for the long-term maintenance of proliferation and cytotoxic activity of the LAK cell clones. The mechanism by which LAK cells kill tumor targets is not known. We here demonstrate that LAK cells and LAK cell clones can produce interferon- and tumor necrosis factor (TNF) when stimulated with an erythroleukemia cell, K562. Five of the six CD3⁺, LAK cell clones tested could be stimulated by K562 cells to produce both interferon- and TNF. However, the ability of the cloned LAK cells to kill K562 cells, as measured in a 4-h ⁵¹Cr-release assay, did not correlate with their ability to produce these cytokines. Furthermore, specific antibodies that neutralize the cytotoxic activity of interferon- and TNF did not inhibit killing of K562 cells by LAK cells as measured with a 4-h cytotoxic assay. The cytostatic and cytotoxic activities of interferon- and TNF for tumor cells are well documented, but these cytolytic activities are slower acting and exhibit their maximum effect after 48–96 h. We here propose that LAK cells kill tumor targets by a combination of cell-to-cell-mediated killing and by the release of slower acting cytostatic/cytotoxic cytokines that can inhibit the growth of tumors some distance from the effector cells.This work is supported in part by grants from the Arizona Disease Research Commission (3364-000000-1-1-AP-6621) and the National Institutes of Health (Grants GM 34121, CA-17094 and CA-23074)     

Recombinant interleukin 2 stimulates in vivo proliferation of adoptively transferred lymphokine-activated killer (LAK) cells

We previously reported that the adoptive transfer of lymphokine-activated killer (LAK) cells plus repetitive injections of recombinant interleukin 2 (IL 2) produced a marked reduction in established pulmonary metastases from a variety of murine sarcomas. The requirement for the exogenous administration of IL 2 prompted a subsequent examination of the role of IL 2 in the in vivo function of transferred LAK cells. The in vivo proliferation and migration patterns of lymphoid cells in C57BL/6 mice were examined after i.v. transfer of LAK cells alone, i.p. injection of IL 2 alone, or the combination of LAK cells and IL 2. A model for in vivo labeling of the DNA of dividing cells was used in which mice were injected with 5-[125I]-iodo-2'-deoxyuridine (125IUdR) and, 20 hr later, their tissues were removed and were counted in a gamma analyzer. A proliferation index (PI) was calculated by dividing the mean cpm of organs of experimentally treated mice by the mean cpm of organs of control mice. In animals given LAK cells alone, the lungs and liver demonstrated little if any uptake of 125IUdR above saline-treated controls (PI = 2.5 and 0.8, respectively, on day 5), whereas the same organs of mice receiving 6000 U of IL 2 alone displayed higher radiolabel incorporation (PI = 7.1 and 5.9, respectively). When mice were given LAK cells plus 6000 U of IL 2, their tissues showed an additional increase in 125IUdR uptake. In the spleen, kidneys, and mesenteric lymph nodes, IL 2 treatment alone (6000 U) produced elevated PI values that were not, however, additionally increased if LAK cells were also administered. To separate the stimulatory effects of IL 2 on host lymphocyte proliferation from similar IL 2 effects on injected LAK cells, these studies were repeated in mice immunosuppressed by 500 rad total body irradiation. Pre-irradiation of the host sufficiently reduced endogenous lymphoid expansion stimulated by IL 2 so as to allow the demonstration that IL 2 also induced the proliferation of the transferred LAK cells. A variety of studies confirmed that the injected LAK cells were actively proliferating in tissues in vivo under the influence of IL 2. Substitution of "normal" LAK cells with fresh and cultured (without IL 2) splenocytes, or irradiated LAK cells did not result in increased 125IUdR uptake in tissues. Histologic studies corroborated the findings of the 125IUdR incorporation assays and revealed extensive lymphoid proliferation in irradiated mice receiving LAK cells plus IL 2.(ABSTRACT TRUNCATED AT 400 WORDS)     

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

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

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.     

Generation of lymphokine-activated killer (LAK) cells and possible implications in autologous bone marrow transplantation--a preliminary report

Lymphokine-activated killer (LAK) cells were generated successfully without mitogen from blood mononuclear cells obtained from 14 patients with varying malignancies and 2 normal donors. Cells from both groups showed a positive cytotoxicity by a 4-hour 51-Cr-release assay against a variety of target cells including natural killer (NK) sensitive K562 myeloid leukemia, NK-resistant Raji lymphoma cell lines, and fresh/cryopreserved leukemia cells from patients refractory to standard chemotherapy but not normal blood cells. Higher cytotoxic activity was obtained with a higher effector:target ratio at 100:1 greater than 50:1 greater than 25:1 (P less than 0.01) in each setting of different targets. Experiments involving cocultures of the LAK cells with either allogeneic (9) or autologous (3) bone marrow cells disclosed no detrimental effect on the committed hemopoietic stem cells by semisolid agar colony forming unit (CFU-GM) assay. The findings suggest that LAK cells may have a potential role for the in vitro purging of the residual leukemic cells from the marrow inoculum prepared for autologous bone marrow transplantation.     

Long-term growth of lymphokine-activated killer (LAK) cells: role of anti-CD3, beta-IL 1, interferon-gamma and -beta

Peripheral blood lymphocytes (PBL) cultured in interleukin 2 (IL 2)-containing medium in conventional tissue culture develop the ability to lyse fresh tumor cells; such cells are referred to as lymphokine-activated killer (LAK) cells. LAK activity peaks by day 5 of culture and declines rapidly thereafter. We studied culture conditions and signals that allow for long-term culture and expansion of cells with LAK activity. By culturing cells at relatively low densities and regularly replenishing medium and recombinant IL 2 (r-IL 2), LAK function is significantly higher as compared with short-term cultures, and remains present for at least 21 days while cell numbers undergo an average 100-fold expansion. By activating these cultures with anti-CD3 (OKT3) monoclonal antibody and r-IL 2, an approximately 1000-fold expansion in the cell number is obtained with maintenance of comparable levels of LAK activity. The exogenous addition of beta interleukin 1 (beta-IL 1), interferon-beta (IFN-beta) or interferon-gamma (IFN-gamma) can augment the lytic activity of cell populations expanded by anti-CD3 plus r-IL 2. These approaches may enable the in vitro generation from individual donors of much greater numbers of LAK cells for adoptive immunotherapy than can now be obtained with the 3 to 5 day in vitro culture systems.     

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.     

Augmentative effect ofNocardia rubra cell-wall skeleton on the induction of human lymphokine-activated killer (LAK) cells by the production of LAK cell helper factor(s)

Summary Nocardia rubra cell wall skeleton (N-CWS) was found to synergistically augment lymphokine-activated killer (LAK) cell generation from human peripheral blood mononuclear cells (PBMC) in the presence of a suboptimal dose of recombinant interleukin-2 (rIL-2). N-CWS increased the number of PBMC expressing IL-2 receptor on their surfaces, and the presence of N-CWS at the early stage of the culture period was essential for the exertion of its augmentative activity on the LAK induction. The predominant phenotype of LAK precursor cells responding to N-CWS and rIL-2 was CD3^– CD16⁺. Culture supernatant from N-CWS-stimulated PBMC was found to act as a substitute for N-CWS in the induction of LAK generation in the presence of rIL-2, suggesting that these cells produced a factor capable of augmenting LAK cell induction (LAK helper factor, LHF). LHF was found to have a molecular mass of 29 kDa by gel filtration, and could also function as a killer helper factor to augment allo-antigen-specific cytotoxic T lymphocyte generation from human peripheral blood T cells as well as murine thymocytes. LHF showed no species specificity, indicating that it is different from IL-4. The enhancing activity of LHF was not neutralized with anti-TNF, anti-IL-1, or anti-IL-1 antibodies. Furthermore, no tumor necrosis factor- (TNF), TNF, IL-1, , IL-2, IL-5, IL-6 or interferon activity was detected in semi-purified LHF during enzyme-linked immunosorbant assay and biological assays. The present findings indicate that LHF produced from N-CWS-stimulated PBMC is a molecule distinct from TNF, TNF, interferon, IL-1, -2, -4, -5, and -6, and suggest that LHF might be a novel lymphokine involved in LAK generation.This work was supported by a Grant-in-aid for Cancer Research from the Ministry of Education, Science, and Culture of Japan