Induction of lymphokine-activated killer activity by interleukin 4 in human lymphocytes preactivated by interleukin 2 in vivo or in vitro.
Carl M. HiguchiJohn A. ThompsonCatherine LindgrenSteven GillisMichael B. WidmerD E KernAlexander Fefer
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In an attempt to augment the generation of human cytotoxic effector cells for potential cancer therapy with interleukin 2 (IL2) and lymphokine-activated killer (LAK) cells, the effect of interleukin 4 (IL4) on LAK cell induction was studied. In normal human peripheral blood lymphocytes (PBL), IL4 does not induce LAK activity and inhibits LAK induction by IL2. However, since lymphocyte activation, such as with antigen or mitogen, can render them responsive to IL4, the ability of IL4 to induce LAK activity in lymphocytes preactivated in vivo or in vitro with IL2 was investigated. PBL obtained from 12 patients with advanced cancer 1 to 3 days after IL2 therapy and from eight healthy control subjects were cultured 4 to 5 days with or without IL4 and/or IL2 and then tested for LAK activity as assessed by lysis of Daudi in a 4-h 51Cr release assay. In normal PBL, IL4 failed to induce LAK activity and consistently inhibited LAK induction by a suboptimal concentration of IL2 (10 units/ml). By contrast, IL4 induced LAK activity in PBL from seven of twelve IL2-treated patients and augmented LAK induction by the suboptimal IL2 in PBL from five of twelve IL2-treated patients. With an optimal LAK-inducing concentration of IL2 (1000 units/ml), IL4 less consistently inhibited LAK induction in normal PBL and had a variable effect upon LAK induction in PBL from IL2-treated patients. IL4 induced LAK activity in PBL obtained from a cancer patient after, but not before, systemic IL2 therapy. Similarly, IL4 induced LAK activity in normal PBL only after they had been preincubated with IL2. Thus, IL4 induces LAK activity in lymphocytes preactivated by IL2 in vivo or in vitro. Fluorescence-activated cell sorting revealed that the LAK activity, whether induced by IL4 or by IL2, was mediated largely by non-T (CD5-) natural killer-like (CD56+) cells. The results suggest a regulatory relationship between IL2 and IL4 in the induction and/or maintenance of LAK activity, which might be exploited to augment the generation of cytotoxic cells for lymphokine-mediated immunotherapy of human cancer.Keywords:
Lymphokine-activated killer cell
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Cryopreserved and thawed lymphocytes can be used instead of fresh lymphocytes to avoid test-to-test variability in studies of fluctuations of natural killer (NK) and lymphokine-activated killer (LAK) activities as a function of time. We investigated the effects of 1-h versus 18-h resting of lymphocytes on their lytic activities, because the process of cryopreservation and thawing decreases NK and LAK activities. Lymphocytes from renal cell cancer patients receiving adoptive immunotherapy were studied. An 18-h versus 1-h resting period led to a significant increase in NK activity but had no significant effect on LAK activity. The presence of 1200 IU/ml interleukin 2 (IL-2) in the medium 1 h prior to and during the cytotoxicity (CTX) assay increased in vivo and in vitro IL-2-induced LAK activities. This phenomenon has been interpreted as IL-2 dependency of effector lymphocytes (J.A. Hank, P.C. Kohler, G. Weil-Hillman, N. Rosenthal, K. H. Moore, B. Storer, D. Minkoff, J. Bradshaw, R. Bechhofer, and P. M. Sondel. Cancer Res., 48: 1965-1971, 1988). We performed kinetic studies to assess the role of effector lymphocyte recruitment in these experiments. LAK activity was tested in the presence or absence of IL-2 during preincubations and CTX assays varying between 0 and 120 min. These kinetic studies showed that effector lymphocyte recruitment indeed contributed to the increased level of LAK activity when IL-2 was added to the CTX assay. A minimal incubation period of 30 min was required to detect recruitment of lymphocytes. Effector lymphocytes could be recruited for periods varying between 90 and greater than 240 min, depending on the lymphocyte donor. We conclude that: (a) in vitro, IL-2-mediated recruitment of lymphocytes due to presence of IL-2 in the CTX assay may lead to an overestimate of the actual LAK activity; and (b) in vivo, prolonged IL-2 infusion after the administration of activated lymphocytes seems warranted in order to recruit maximal levels of effector lymphocytes with LAK activity.
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Culture of human peripheral blood leukocytes with interleukin 2 (IL-2) stimulates their differentiation into lymphokine-activated killer (LAK) cells, with a broad range of cytotoxicity against fresh tumor cells and tumor cell lines (Grimm et al., J. Exp. Med., 155: 1823-1841, 1982). We chose to utilize a molecular approach to determine whether IL-2 stimulates the expression of cytokine genes by the mixed cell population which may be involved in the generation or regulation of lytic activity. Northern blot analysis performed with total cellular RNA from LAK cells cultured for varying periods of time with IL-2 revealed that the genes which code for cytokines [interleukin 1 (IL-1)alpha and beta, gamma-interferon, tumor necrosis factor alpha, and lymphotoxin] were not spontaneously expressed. As soon as 2 h after IL-2 treatment, IL-1 alpha and IL-1 beta mRNAs were expressed. Both nonadherent and adherent populations of LAK cells express IL-1 beta mRNA; however, the adherent population produced more IL-1 beta mRNA and maintained its expression for a prolonged period of time. Other cytokine mRNAs (gamma-interferon, tumor necrosis factor alpha, and lymphotoxin) were expressed later than the IL-1 mRNAs with maximal levels between Days 2 through 7. Our results indicate that LAK cell populations can generate a variety of cytokines which may be involved in the generation of lytic activity.
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Peripheral blood lymphocytes from 42 patients and peritoneal cavity lymphocytes from eight patients with advanced ovarian carcinoma were tested for lymphokine-activated killer lymphocyte cytotoxicity against several ovarian carcinoma lines before and after exposure to recombinant interleukin-2 in vitro for 3-5 days. Only four of 42 (9.5%) of peripheral blood lymphocytes and zero of eight (0%) of peritoneal cavity lymphocytes had spontaneous cytotoxicity (greater than 20%) against the ovarian carcinoma lines. After in vitro exposure to recombinant interleukin-2, 41 of 42 (98%) of patients' blood lymphocytes showed a two- to fivefold increase in cytotoxicity against K562 (a lymphoblastoid human target), and 40 of 42 (95%) demonstrated lymphokine-activated killer cytotoxicity (greater than 20%) to the ovarian carcinoma lines. Lymphokine-activated killer activity against fresh allogeneic cell lines was variable, although most patients' peripheral blood lymphocytes (70%) had significant cytotoxicity. By contrast, incubation of patients' peritoneal cavity lymphocytes with recombinant interleukin-2 in vitro did not result in the generation of lymphokine-activated killer cell activity against K562 or ovarian cell lines. Peritoneal lymphocytes did produce lymphokine-activated killer cells in the presence of OK432 in half of the patients tested. The presence of autologous serum during recombinant interleukin-2 activation with blood lymphocytes had an augmentative effect on the resulting lymphokine-activated killer cytotoxicity in two of 20 patients, a suppressive effect in four of 20, and no effect in the other 14 of 20 patients tested.(ABSTRACT TRUNCATED AT 250 WORDS)
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The lymphocyte-mediated cytotoxicity and cytokine production of patients under maintenance haemodialysis were studied. The patients were subdivided into two groups; 27 patients receiving haemodialysis treatment for up to 3 years (group 1) and 27 patients for longer than 10 years (group 2). Twenty-six healthy volunteers (group 3) were used as controls. The immunological parameters assayed were NK cell and lymphokine-activated killer (LAK) cell activities, and production of both interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha by either peripheral blood mononuclear cells (PBMC) cultured with IL-2 or LAK cells. In group 1 both NK- and LAK-cell-mediated cyto-toxicities were significantly suppressed as compared to those in group 3. The titre of IFN produced by LAK cells was also less than that in group 3. On the contrary there were no significant differences in cytokine production by PBMC in all three groups. Percentage of the cases with suppressed cytokine production seemed higher in group 1 than group 3. In addition there was a close correlation between the value of IFN-gamma and of TNF-alpha produced by PBMC in the patients on haemodialysis. Both the type of dialysis membranes used and the primary diseases did not significantly influence the results in group 1 and group 2. From these results it could be speculated that there are more patients with impaired immune function in group 1 than in group 3. In contrast there was no significant difference in the immune function tested between group 2 and group 3. Our results indicate the need for a study on time kinetics concerning the change in suppressed immune function of the patients and their clinical courses shortly after the beginning of haemodialysis.
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Cryopreserved and thawed lymphocytes can be used instead of fresh lymphocytes to avoid test-to-test variability in studies of fluctuations of natural killer (NK) and lymphokine-activated killer (LAK) activities as a function of time. We investigated the effects of 1-h versus 18-h resting of lymphocytes on their lytic activities, because the process of cryopreservation and thawing decreases NK and LAK activities. Lymphocytes from renal cell cancer patients receiving adoptive immunotherapy were studied. An 18-h versus 1-h resting period led to a significant increase in NK activity but had no significant effect on LAK activity. The presence of 1200 IU/ml interleukin 2 (IL-2) in the medium 1 h prior to and during the cytotoxicity (CTX) assay increased in vivo and in vitro IL-2-induced LAK activities. This phenomenon has been interpreted as IL-2 dependency of effector lymphocytes (J.A. Hank, P.C. Kohler, G. Weil-Hillman, N. Rosenthal, K. H. Moore, B. Storer, D. Minkoff, J. Bradshaw, R. Bechhofer, and P. M. Sondel. Cancer Res., 48: 1965-1971, 1988). We performed kinetic studies to assess the role of effector lymphocyte recruitment in these experiments. LAK activity was tested in the presence or absence of IL-2 during preincubations and CTX assays varying between 0 and 120 min. These kinetic studies showed that effector lymphocyte recruitment indeed contributed to the increased level of LAK activity when IL-2 was added to the CTX assay. A minimal incubation period of 30 min was required to detect recruitment of lymphocytes. Effector lymphocytes could be recruited for periods varying between 90 and greater than 240 min, depending on the lymphocyte donor. We conclude that: (a) in vitro, IL-2-mediated recruitment of lymphocytes due to presence of IL-2 in the CTX assay may lead to an overestimate of the actual LAK activity; and (b) in vivo, prolonged IL-2 infusion after the administration of activated lymphocytes seems warranted in order to recruit maximal levels of effector lymphocytes with LAK activity.
Lymphokine-activated killer cell
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In an attempt to augment the generation of human cytotoxic effector cells for potential cancer therapy with interleukin 2 (IL2) and lymphokine-activated killer (LAK) cells, the effect of interleukin 4 (IL4) on LAK cell induction was studied. In normal human peripheral blood lymphocytes (PBL), IL4 does not induce LAK activity and inhibits LAK induction by IL2. However, since lymphocyte activation, such as with antigen or mitogen, can render them responsive to IL4, the ability of IL4 to induce LAK activity in lymphocytes preactivated in vivo or in vitro with IL2 was investigated. PBL obtained from 12 patients with advanced cancer 1 to 3 days after IL2 therapy and from eight healthy control subjects were cultured 4 to 5 days with or without IL4 and/or IL2 and then tested for LAK activity as assessed by lysis of Daudi in a 4-h 51Cr release assay. In normal PBL, IL4 failed to induce LAK activity and consistently inhibited LAK induction by a suboptimal concentration of IL2 (10 units/ml). By contrast, IL4 induced LAK activity in PBL from seven of twelve IL2-treated patients and augmented LAK induction by the suboptimal IL2 in PBL from five of twelve IL2-treated patients. With an optimal LAK-inducing concentration of IL2 (1000 units/ml), IL4 less consistently inhibited LAK induction in normal PBL and had a variable effect upon LAK induction in PBL from IL2-treated patients. IL4 induced LAK activity in PBL obtained from a cancer patient after, but not before, systemic IL2 therapy. Similarly, IL4 induced LAK activity in normal PBL only after they had been preincubated with IL2. Thus, IL4 induces LAK activity in lymphocytes preactivated by IL2 in vivo or in vitro. Fluorescence-activated cell sorting revealed that the LAK activity, whether induced by IL4 or by IL2, was mediated largely by non-T (CD5-) natural killer-like (CD56+) cells. The results suggest a regulatory relationship between IL2 and IL4 in the induction and/or maintenance of LAK activity, which might be exploited to augment the generation of cytotoxic cells for lymphokine-mediated immunotherapy of human cancer.
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Lymphokine-activated killer cells were generated from peripheral blood mononuclear cells of 33 patients with liver tumors (benign, 6; primary malignant, 10; metastatic, 17) and 10 healthy individuals. Although peripheral blood mononuclear cell yield was significantly lower (p < 0.01) in patients with hepatocellular carcinoma or with metastatic colorectal cancer, natural killer activity in the peripheral blood mononuclear cell fraction was comparable in all groups tested. Optimal lymphokine-activated killer activity was demonstrated after 9 to 12 days of culture in recombinant interleukin 2. Lymphokine-activated killer activity, interleukin 2-induced lymphocyte proliferation and total lytic activity generated per culture in all patient groups studied were similar to those of normal control cells cultured under the same conditions. These in vitro data demonstrate the feasibility of obtaining lymphokine-activated killer cells from the blood of patients with liver tumors and provide a rationale for the future use of lymphokine-activated killer cells in adoptive immunotherapy of patients with primary and metastatic hepatic neoplasms.
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To test the hypothesis that reduced lymphocyte transformation in response to PHA in chronic hepatitis B virus infection might be due to deficient lymphokine production, lymphocyte transformation was measured in the presence or absence of exogenous interleukin 1, interleukin 2 or both, or, as a source of mixed lymphokines, supernatants from mixed lymphocyte reactions. The response to PHA was significantly impaired in patients compared to controls, but was not corrected by interleukin 1, interleukin 2 or supernatant from mixed lymphocyte reactions over a wide range of concentrations. Variation of the proportion of monocytes in culture or the addition of indomethacin had no effect on lymphocyte transformation. Thus, reduced lymphocyte proliferation in response to PHA in patients with chronic hepatitis B virus infection cannot be attributed to deficient lymphokine production or to active suppression by monocytes or prostaglandins and a direct role for the hepatitis B virus or a viral product is under investigation.
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Peritoneal cavity
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Lytic cycle
Lymphokine-activated killer cell
Aldesleukin
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