Recombinant interleukin-2 and lymphokine-activated killer cells in renal cancer patients: II. characterization of cells cultured ex vivo and their contribution to the in vivo immunomodulation

Summary Burkitt lymphoma (BL) lines can be grouped according to phenotypic characteristics. Group I cells exhibit the phenotype of resting B cells and grow as single cells. Such lines can be Epstein-Barr-virus(EBV)-negative or -positive. Group II and group III cells are always EBV-positive, they express B cell activation markers, grow in aggregates and resemble in varying degrees lymphoblastoid cell lines (LCL). We studied three groups of BL lines for their capacity to interact with allogeneic lymphocytes. The results showed that as long as the lines have the group I phenotype, they do not stimulate allogeneic T lymphocytes irrespective whether they carry the EBV genome. The group II and III cells are stimulatory. Generally there was no correlation between sensitivity to lymphocyte-mediated lysis and the phenotype of the lines. In one set of lines, the group I cells had higher sensitivity to both natural killer and lymphokine-activated killer effectors compared to the group II or III lines. However, such correlation could not be seen with the other two sets of lines. Among the phenotypic features investigated, expression of the adhesion molecules LFA-1 and LFA-3 correlated with the tendency for cell aggregation.     

The augmentation of lymphokine-activated killer activity following pulsing of human peripheral blood mononuclear cells with recombinant human interleukin-2

Summary The short-term exposure of peripheral blood mononuclear cells (PBMC) to recombinant human interleukin-2 (rhIL-2) at 37°C leads to the generation of lymphokine-activated killer (LAK) activity similar in magnitude to that obtained by the exposure of PBMC to rhIL-2 continuously for 3–5 days. In order to investigate whether the required signal for LAK induction occurred during the short exposure to rhIL-2 or at a later point in the induction phase, PBMC were exposed to rhIL-2 for 1 h at 4°C and then exposed to a low-pH wash to remove bound IL-2 from its receptor. PBMC treated in such a way showed increased LAK activity and proliferation compared to cells exposed to rhIL-2 alone. Expression of the p55 () subunit of the IL-2 receptor was also increased. In order to cause the augmentation, a lowering of the pH below 4.0 was necessary, and exposure of PBMC to low pH alone (in the absence of rhIL-2) failed to cause activation. Another relevant feature was a transient increase in the expression of the p75 subunit of the IL-2 receptor ( chain) immediately following the exposure to low pH and the release of interferon , tumour necrosis factor and IL-6; activation was blocked by the inclusion of neutralising antisera raised against rhIL-2 and interferon , thus demonstrating that the endogenous release of these cytokines is important for activation.     

Cytotoxic cell function and phenotypic analysis of peripheral blood mononuclear cells in cancer patients treated with low-dose interleukin-2 and mitomycin C

We previously found that the ability of peripheral blood mononuclear cells (PBM) of cancer patients to generate lymphokine-activated killer (LAK) cells became remarkably augmented after mitomycin C administration. On the basis of the clinical finding, we designed a treatment regimen comprised of 12 mg/m² mitomycin C i. v. on day 1 and 700 U/m² recombinant interleukin-2 (IL-2) i.v. every 12 h from day 4 through day 8. Of 25 patients with advanced carcinoma, 9 had a partial response and 3 had a minor response. Cytotoxic cell function, including natural killer activity, lymphokine-activated killer (LAK) activity, and the ability to generate LAK cells, and lymphocyte subsets in PBM was measured 1 day before and after either the first or second course of this therapy. The relationship between these parameters and the clinical antitumor response to this treatment was examined. Although the cytotoxic activities were significantly augmented after either the first or second treatment course, no positive correlation was observed between the changes in these cytotoxic activities and the clinical response to this therapy, when patients who either showed a partial response or whose disease remission was partial or minor were defined as responders. Further, phenotypic analysis showed a significant increase in CD2⁺, CD3⁺ CD4⁺ and CD4⁺Leu8^– cells after the firs course, and CD25⁺ cells after either the first or second course of this treatment. The precentages of CD2⁺ and CD25⁺ cells were significantly elevated only in responders but not in nonresponders, suggesting the increase in these subsets was related to clinical response.     

Assessment of the functional activity of lymphokine-activated killer cells in healthy donors and in patients with interleukin-2-dependent immunodeficiency

The methodological aspects of Lak-cells functional activity test were determined. NK-resistance of P-815 culture cells and NK-sensitive K-562 cells were used as target cells. The highest cytotoxicity level was registered at the 5-th day of culturing MNC with rIL-2 at the final concentration (100 U/ml). The method developed was used for testing healthy donors group and group of patients with IL-2 dependent immunodeficiency (ophthalmic herpes). Data obtained show the significant decrease of LAK-cells functional activity of patients in comparison with healthy donors. Thus, the developed method is recommended for complex estimation of immune status of patients with IL-2-dependent immuno deficiency.     

Cryopreservation-induced enhancement of interleukin-2 production in human peripheral blood mononuclear cells.

To better understand the effects of cryopreservation on various immunocompetent cell functions, we have examined the interleukin-2 (IL-2)-producing activities of frozen mononuclear cells (MNCs) from healthy subjects. The mechanisms responsible for the observed effects were also analyzed. Both the unfractionated and monocyte-depleted, frozen MNCs produced significantly larger quantities of IL-2 than fresh cells. Similar to freezing, L-leucine methyl ester (Leu-OMe) treatment (to eliminate IL-1 and prostaglandin E-2 (PGE-2)-secreting cells) also increased the IL-2-producing activities of fresh cells, but freezing no longer enhanced the production of IL-2 by Leu-OMe-treated cells, suggesting that (1) both the freezing process and Leu-OMe treatment have similar effects on IL-2 production, (2) the increased IL-2 secretion by frozen MNCs is independent of IL-1, and (3) inactivation of PGE-2-secreting cells during the freezing procedure is responsible for increased IL-2 secretion. Elimination of CD8+ T cells (putative suppressor cells) from MNCs has also resulted in the production of increased amounts of IL-2 by fresh cells, and again, freezing did not further enhance the IL-2-secreting activities of MNCs, that are devoid of CD8+ T cells. This confirms that the increased IL-2 production is due to the inactivation of immuno-down-regulatory cells. The results provide further evidence that the lack of active, suppressor T cells, monocytes, and increased IL-1 and -2 production may be responsible for the previously reported enhanced immunoglobulin-producing abilities of cryopreserved cells from healthy subjects and from patients with lung cancer.     

Chemiluminescence in peripheral blood mononuclear cells of solid tumor cancer patients

Summary Levels of chemiluminescence were measured in peripheral blood mononuclear cells (PBMC) from normal subjects and from solid tumor cancer patients. Patients with advanced malignant disease were found to have significantly elevated baseline chemiluminescence activity in their resting PBMC as compared to normal subjects or to cancer patients with, at most, minimum residual disease. Patients with either advanced disease or minimum residual disease, however, were found to exhibit significantly elevated activation of chemiluminescence by treatment of cells with phorbol myristic acetate (PMA). Treatment of surgically resected stage I lung cancer patients with Freund's complete adjuvant alone or emulsified with extracted lung cancer antigens was found to elevate chemiluminescence levels in patient PBMC. Serum from those vaccinated patients was found to elevate chemiluminescence levels of resting PBMC from normal subjects. That serum activity did net correlate with levels of immune complexes measurable in the Clq or Raji cell assay.     

Use of human leukocyte antigen-mismatched allogeneic lymphokine-activated killer cells and interleukin-2 in the adoptive immunotherapy of patients with malignancies.

Clinical effects and side effects were investigated in the adoptive immunotherapy of patients bearing malignant diseases using human leukocyte antigen (HLA)-mismatched allogeneic lymphokine-activated killer (LAK) cells. Allogeneic LAK cells were induced from peripheral blood lymphocytes (PBL) of healthy donors with the same blood types as those of patients. Recently we succeeded in increasing the proliferation rate and enhancing the cytotoxic activity of LAK cells by means of initial stimulation with pokeweed mitogen (PWM, PWM-LAK cells). Five of 12 patients applied in the adoptive immunotherapy showed clinical effects such as partial or complete regression of pulmonary metastases and pleural effusion. All pulmonary metastatic lesions were eliminated in one case by this adoptive immunotherapy combined with chemotherapy. Toxic effects were chillness, fever and general fatigue which were reversible, and no allergic side effects occurred even though allogeneic LAK cells were injected frequently. In the patients who received more than 10(11) of allogeneic LAK cells, anti-HLA class I antibodies appeared without any evidence of autoantibody. However, immunological side effects were never experienced after injection of allogeneic LAK cells even when the anti-HLA class I antibodies existed in the patients; this phenomenon suggests the safety of the adoptive immunotherapy using allogeneic LAK cells. Taken together, allogeneic LAK cells could be considered as alternative therapy for patients with malignancies who could not supply sufficient materials of autologous LAK cells. Recently, LAK cells, particularly PWM-LAK cells were found to obtain significantly potent and prompt lectin-dependent cell-mediated cytotoxicity (LDCC). All tumor cells confluent in microtest plate well could be annihilated by PWM-LAK cells plus PWM less than 8 hours. New immunotherapy using PWM-LAK cells or lectin-stimulated LAK cells with PWM or other lectins is discussed.     

Immunomodulatory effects of systemic low-dose recombinant interleukin-2 and lymphokine-activated killer cells in humans

Summary The adoptive immunotherapy of human cancer using lymphokine-activated killer (LAK) cells in combination with high-dose systemic recombinant interleukin-2 (rIL-2) has been associated with global changes in several hematological and immunological parameters while imposing profound toxicity on patients. We have evaluated an alternative LAK cell therapy utilizing low-dose systemic rIL-2 in 27 consecutive patients with metastatic cancer. We report that the administration of systemic low-dose rIL-2 is also characterized by significant changes in immunological and hematological parameters, which are qualitatively similar to those induced by high-dose rIL-2. Low-dose systemic rIL-2, given by i.v. bolus, is cleared to baseline levels within 240 min of administration. The induction of lymphocytosis and eosinophilia, which has characterized other protocols, is also a feature of this protocol. In addition, low-dose systemic rIL-2/LAK cell immunotherapy results in increased peripheral blood mononuclear cell (PBMC) expression of T-cell activation markers such as OKIa, OKT10 and IL-2 receptor. PBMC sampled approximately 100 h after the final infusion of LAK cells demonstrated a statistically significant increase in their ability to kill natural killer (NK)-sensitive and NK-resistent cell lines such as K562 and Daudi compared to baseline values (P <.05). These data suggest that rIL-2-based immunotherapy using low-dose rIL-2 is capable of inducing quantitative hematological and immunological changes while (in combination with LAK cells) retaining the ability to mediate tumor regressionin vivo. Dr. Eberlein was a recipient of an American Cancer Society Career Development Award. This work is supported in part by NIH Grant CA-40555 and the Clinical Research Center Grant 20-9299     

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.     

Cytomegalovirus infection of peripheral blood mononuclear cells: effects on interleukin-1 and -2 production and responsiveness.

Cytomegalovirus suppresses the proliferative response of peripheral blood mononuclear cells to phytohemagglutinin. In these experiments, we identified which mononuclear cell subpopulation might be responsible for the suppression. We found that prior infection of either lymphocytes or monocytes followed by reconstitution with monocytes or lymphocytes, respectively, would abrogate the proliferative response in a subsequent culture with phytohemagglutinin. Infection of either cell type also reduced both the production of interleukin-1 (IL-1) and IL-2 and the proliferative response to exogenously supplied IL-1 or IL-2. We did not find evidence for an IL-2 antagonist. These experiments suggest that cytomegalovirus causes a metabolic derangement in lymphocytes and monocytes and impairs their ability both to produce and to respond to physiological mediators of the immune response.     

The natural killer and fungicidal activity of the peripheral blood mononuclear cells in candidiasis

The activity of natural killers and the capacity of peripheral blood mononuclears for the extracellular killing of Candida cells in patients with different forms of candidiasis has been determined in the radiometric test with the use of targets labeled with 3H-uridine RNAase and in the fungicidal test. On the basis of the data thus obtained the conclusion has been made that in candidiasis changes in the activity of natural killers and fungicidal capacity depend on the severity of the disease and the stage of the process. Linear correlation between the activity of natural killers and fungicidal capacity in candidiasis patients at the stage of exacerbation and in healthy persons is absent.     

Regulation of Staphylococcus aureus-mediated activation of interleukin-18 in peripheral blood mononuclear cells.

Bacteria and bacterial antigens strongly induce cytokine secretion by peripheral blood leukocytes and thereby initiate an inflammatory cascade with potentially deleterious consequences for the host. The present study focussed on receptors and signal transduction pathways involved in activation of interleukin (IL)-18 by heat-inactivated Gram-positive Staphylococcus aureus Cowan strain I (SAC). Similarly to IL-12/IL-12p40, IL-10 and IFN-gamma, SAC dose-dependently activated IL-18. Secretion of IL-18 was independent of functional activity of IL-10, IL-12 or IFN-gamma. Lipoteichoic acid (LTA), a structural component of SAC, was not sufficient for activation of IL-18, while it dose-dependently induced IL-10. In contrast to IL-12, blockade of CD14 only partially diminished secretion of IL-18 and did not affect secretion of IL-10, suggesting involvement of other receptors (e.g., Toll-like receptors) in SAC responses. Further down-stream however, secretion of IL-10, IL-12 and IL-18 was uniformly inhibited by blockade of G-protein-mediated kinase activation by mastoparan. Secretion of IL-18 required phosphatidylinositol-3'-kinase, and secretion of IL-12 phosphotyrosine kinase activity. The data demonstrate that SAC potently activates secretion of IL-18 by peripheral blood mononuclear cells with differential involvement of cell-surface receptors and signal transduction pathways as compared to other natural killer- and T cell-promoting cytokines.     

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

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

Induction of lymphokine-activated killer cells from rat thymocytes using recombinant human interleukin-2

Summary Using a 4-h ⁵¹Cr release assay, we observed that thymocytes from Fischer strain rats incubated with recombinant human interleukin-2 (rhIL-2) developed cytotoxicity to YAC-1 lymphoma, 9L-glioma, and B-16 melanoma cells (effector/target ratio =25/1). Induction of the lymphokine-activated killer (LAK) cells was as follows: (1) when 5×10⁶/ml thymocytes were cultured with various concentrations of rhIL-2 (50, 125, 250, 500, or 1,000 units/ml) for 4 days, no cell proliferation was observed at any concentration. However, the LAK cells showed significant cytotoxicity toward all tumor cells at more than 50 units/ml. (2) When 5×10⁶/ml thymocytes were cultured for 1 to 6 days with 250 units/ml of rhIL-2, the harvested cell count decreased markedly after the 2nd day. The cytotoxicity of all the tumor cells became significant after the 2nd day, with peak activity on the 4th day. In rat splenocytes, on the other hand, the LAK cells could not be identified because rat splenocytes developed nonspecific cytotoxicity in medium containing fetal calf serum without adding rhIL-2.     

Loco-regional immunotherapy with recombinant interleukin-2 and adherent lymphokine-activated killer cells (A-LAK) in recurrent glioblastoma patients

Nine patients with recurrent glioblastoma were given autologous adherent lymphokine-activated killer (A-LAK) cells and interleukin-2 (IL-2) administered directly into the tumor cavity through an Ommaya tube placed during surgery/biopsy. The immunotherapy was well tolerated and the response rate was 33% (one complete response, two partial responses, four with stable disease and two with progressive disease). However, survival 18 months from initial diagnosis did not differ from that reported in the literature for patients treated conventionally. Serial determinations of IL-2 in the tumor cavity during the course of treatment revealed that IL-2 concentrations were sufficient to maintain lymphocyte activation. Since steroid medication was discontinued during treatment and A-LAK cells have greater antitumor activity than standard LAK cells, other factors are discussed that might explain the limited results.     

Activation of eosinophils in cancer patients treated with IL-2 and IL-2-generated lymphokine-activated killer cells

Of 16 patients, a total of 13 who received IL-2 and autologous IL-2-generated lymphokine-activated killer LAK cells developed eosinophilia late during the course of treatment. To understand the direct or indirect effects of IL-2 on eosinophils, the physical and functional characteristics of the late-treatment eosinophils were compared to those of early-treatment and control eosinophils. Late-treatment eosinophils differed from early-treatment and control eosinophils in the following respects: they had somewhat reduced density, hypersegmented nuclei, eosinophil cationic protein converted from the storage form to the secretory form, and a greater than 200% increased ability to kill larvae of Schistosoma mansoni by an antibody-dependent mechanism (cytotoxic function). In vitro, IL-2 (1000 U/ml in medium as used to culture LAK cells) did not affect the cytotoxic function of eosinophils from cancer patients or from control subjects. However, LAK cell-conditioned medium enhanced the cytotoxic function of eosinophils from early-treatment cancer patients and from normal subjects by greater than 150%. Thus, eosinophils late in the course of IL-2/LAK cell treatment undergo physical changes and become functionally activated. The involvement of IL-2 in these changes is probably indirect, as an inducer of factors that enhance eosinophil function.     

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.     

Use of human leukocyte antigen-mismatched allogeneic lymphokine-activated killer cells and interleukin-2 in the adoptive immunotherapy of patients with malignancies

Clinical effects and side effects were studied in the adoptive immunotherapy of patients bearing malignant diseases using human leukocyte antigen (HLA)-mismatched allogeneic lymphokine-activated killer (LAK) cells. Allogeneic LAK cells were induced from peripheral blood lymphocytes (PBL) of normal donors by means of initial stimulation with pokeweed mitogen (PWM). Six of 15 patients applied in the adoptive immunotherapy showed clinical effects such as partial or complete regression of pulmonary metastasis, pleural effusion and primary tumor. All pulmonary metastatic lesions were eliminated in one case by this adoptive immunotherapy combined with chemotherapy. Generally toxic effects were chillness, fever and general fatigue which were reversible, and no allergic side effects occurred even though allogeneic LAK cells were injected frequently except one patient who showed preshock like symptom accompanied with leukocytopenia and continuous hypotension immediately after infusion but was finally rescued. In the patients who received more than 10¹¹ of allogeneic LAK cells, anti-HLA class I antibodies appeared without any evidence of autoantibody. However, immunological side effects were never experienced after injection of allogeneic LAK cells even when the anti-HLA class I antibodies appeared in the patients. Taken together, allogeneic LAK cells could be considered as alternative therapy for patients with malignancies who could not supply sufficient materials of autologous LAK cells.Abbreviations PWM pokeweed mitogen - LAK lymphokine-activated killer - IL-2 interleukin 2 - PEL peripheral blood lymphocytes - TIL tumor-infiltrating lymphocytes - GVHD graft-versus-host disease - HLA human leukocyte antigen