Lymphokine-activated Killer Precursor Research Articles

Lymphokine-activated killer (LAK) cell generation from peripheral blood stem cells by in vitro incubation with low-dose interleukin-2 plus granulocyte-macrophage colony-stimulating factor.

Previous reports have demonstrated granulocyte-macrophage colony-stimulating factor (GM-CSF)-mediated enhancement of lymphokine activated killer (LAK) cell function. Based on these studies we have developed a model of LAK cell generation from peripheral blood stem cells (PBSC) from cancer patients by in vitro incubation with low-dose interleukin-2 (IL-2) + GM-CSF. PBSC from seven patients were incubated for 48 h at 37 degrees C in serum-free culture medium supplemented with IL-2 at increasing concentrations (10, 100 or 1000 IU/ml) in the presence or absence of 10 IU/ml GM-CSF. LAK activity generated in cultures with 10 IU/ml IL-2 + GM-CSF was significantly higher than that generated by 10 IU/ml IL-2 and did not differ from LAK generation at optimal concentrations of IL-2 (100 and 1000 IU/ml). PBSC from five additional patients were incubated with low-dose IL-2 + GM-CSF after sequential depletion of the CD4+ and CD8+ T cell subsets. LAK activity was significantly reduced by depletion of both CD4+ and CD8+ T cells and almost completely abolished after depletion of both subsets, suggesting that T cells and not NK cells are the main LAK precursors in this model. Six patients have received two courses of LAK cells generated in vitro by low-dose IL-2 + GM-CSF on day +1 and +8 after PBSC transplant in combination with GM-CSF and IL-2 administration in vivo. The mean LAK activity in peripheral blood of these patients dramatically increased immediately after transplant from a mean of 10% to 43.2% on day +2 and remained increased during the period studied. These results are encouraging and suggest that the administration of in vitro generated LAK cells early after transplant may have a role in the control of minimal residual disease.

Mechanism of exercise-induced augmentation of lymphokine activated killer (LAK) cell activity in the horse.

Intense exercise affects various parameters of the immune system. The overall effect of exercise on immune function is dependent upon the physical condition of the subject, the intensity and duration of the exercise period, and the immune parameter assessed. Unconditioned horses subjected to a single bout of intensive exercise exhibit multiple alterations in immune function, including an augmentation of lymphokine activated killer (LAK) cell function. This increase in LAK cell activity is not due to an increase in circulating LAK precursors. While peripheral blood mononuclear cells from exercising horses exhibit greater responsiveness to IL-2, this is not due to an increase in IL-2 receptor expression. LAK cell generation in vitro is augmented by those catecholamines and neuropeptides which are produced during exercise, suggesting a direct effect of these compounds on LAK cell generation at a step post IL-2 receptor binding.

Partial night sleep deprivation reduces natural killer and celhdar immune responses in humans

Prolonged and severe sleep deprivation is associated with alterations of natural and cellular immune function. To determine whether alterations of immune function also occur after even a modest loss of sleep, the effects of early-night partial sleep deprivation on circulating numbers of white blood cells, natural killer (NK) cell number and cytotoxicity, lymphokine-activated killer (LAK) cell number and activity, and stimulated interleukin-2 (IL-2) production were studied in 42 medically and psychiatrically healthy male volunteers. After a night of sleep deprivation between 10 P.M. and 3 A.M., a reduction of natural immune responses as measured by NK cell activity, NK activity per number of NK cells, LAK activity, and LAK activity per number of LAK precursors (CD16,56, CD25) was found. In addition, concanavalin A-stimulated IL-2 production was suppressed after sleep deprivation due to changes in both adherent and nonadherent cell populations. After a night of recovery sleep, NK activity returned to baseline levels and IL-2 production remained suppressed. These data implicate sleep in the modulation of immunity and demonstrate that even a modest disturbance of sleep produces a reduction of natural immune responses and T cell cytokine production.

Isolation of Human NK Cells and Generation of LAK Activity

This unit describes the preparation of natural killer (NK) cells from normal human peripheral blood and their incubation in vitro in the presence of interleukin 2 (IL-2) to yield lymphokine-activated killer (LAK) cells. A protocol is presented for isolating highly purified NK cell populations from PBMC, and another method presents steps for generating LAK cells from these purified NK cells via IL-2 stimulation. An alternate protocol describes the generation of LAK cells directly from whole, unseparated PBMC preparations instead of purified NK populations. The caveat with the alternate protocol is that LAK activity generated in this manner represents the total cytotoxic potential of all LAK precursor cells--i.e., all those PBMC that are capable of responding to IL-2 by up-regulation of cytotoxicity against NK-resistant targets. In PBMC, LAK precursor cells are found among subpopulations of both NK cells and T lymphocytes.

Lamina propria mononuclear cells express and respond to interleukin-2 differently in Crohn's disease and ulcerative colitis.

Lymphokine-activated killer (LAK) cell activity and messenger RNA (mRNA) expression for interleukin-2 (IL-2) were analyzed using lamina propria mononuclear cells (LPMC) in inflammatory bowel disease patients. Compared with control LPMC, Crohn's disease (CD) LPMC exhibited significantly higher levels of LAK cell activity, whereas ulcerative colitis (UC) cells showed significantly lower levels of cytolytic activity with a difference in the frequency of CD3+, CD56+ and CD3+, CD56+ LAK precursor cells. After incubation with IL-2, the proportion of CD3+, CD56+ lymphocytes continued to be higher in CD cultures and substantially lower in UC cultures. Freshly isolated CD LPMC exhibited significantly higher levels of IL-2 mRNA than controls. However, no significant difference was observed between UC and control cells. The level of IL-2 expression or responsiveness to IL-2 may be responsible for different mucosal immune reactivity between CD and UC patients.

Effects of Ultraviolet-B Irradiation on Human LAK and NK Cytotoxic Activity

In vitro ultraviolet-B (UVB) irradiation of murine and rodent bone marrow cells prevents GVHD without compromising engraftment while inducing tolerance to donor-type allografts. In anticipation of clinical trials of UVB-modified bone marrow grafts, we studied the in vitro effects of UVB irradiation (50-300 J/m 2) on human natural killer and lymphokine activated killer cells since both types of cells influence the development of GVHD and graft-versus-tumor effect. Interleukin-2-activated and untreated human lymphocytes were used as effectors in a 51Cr release cytotoxic assay against various tumor cell lines as targets. NK-mediated lysis of K562 targets was decreased by UVB irradiation of the effector cells in a dose-dependent manner. FACS analysis of CD16 + and CD56 + cells 24 hr after UVB exposure showed a UVB-dose-dependent decrease in the number of cells expressing these surface markers. UVB irradiation of lymphocytes prior to activation with high-dose IL-2 resulted in a range of 20- to 89-fold decrease in LAK precursors as measured by limiting dilution analysis using the LAK-sensitive cell line HL60. In contrast, the LAK activity of lymphocytes that had been stimulated in vitro with high-dose IL-2 prior to UVB irradiation was preserved when assayed immediately after UVB modulation; however, there was a significant decrease in lytic activity (with most samples tested) when the assay was performed 24 hr after UVB exposure. It appears that the lymphocyte response to UVB modification is dose dependent, with some cell types displaying higher sensitivity to UVB irradiation than others. These findings suggest that prevention of GVHD by UVB is due, in part, to inhibition of NK activity, and may offer a new strategy to augment the graft versus leukemia effect of UVB-modified bone marrow grafts in clinical transplantation.

Chemotherapy-induced modulation of natural killer and lymphokine-activated killer cell activity in euthymic and athymic mice.

Combinations of chemotherapy and interleukin-2 (IL-2) aimed at improving therapeutic efficacy in cancer patients have generally proved disappointing. Although chemotherapy blocks tumor growth and sometimes boosts immune functions, most drugs are immunosuppressive, at least transiently. Therefore, it is reasonable to assume that maximal exploitation of the immunostimulatory and antitumor activity of both modalities requires careful coordination of chemotherapy and IL-2 timing. We analyzed the temporal effect of 5-fluorouracil (5-FU, 100-120 mg/kg), cyclophosphamide (CY, 100 mg/kg), Adriamycin (8 mg/kg) and dacarbazine (100 mg/kg) on the activation of natural killer/lymphokine-activated killer (NK/LAK) cells by IL-2 in several strains of euthymic mice and in athymic nude mice. Following in vivo or in vitro exposure to IL-2 1-15 days after chemotherapy, the total lytic activity of the spleen and the number of LAK precursors (LAK-p) were measured. In euthymic mice injected with IL-2 (5 x 10(4) Cetus units twice daily for 4-5 days), 5-FU augmented (up to 37-fold, days 1-9) and CY reduced (up to day 6) LAK activity, as compared with that in the IL-2 control. In bulk cultures containing IL-2 (1000 CU/ml, 3-4 days), both 5-FU and CY reduced LAK activity of euthymic mice splenocytes for up to 6 days after chemotherapy, which was followed on day 9 by full recovery. In splenocytes of nude mice, 5-FU increased and CY diminished LAK activation in bulk cultures, starting 3 days after chemotherapy. In athymic mice, 5-FU markedly augmented the total number of LAK-p/spleen (up to 30-fold, days 3-9), as determined by limiting-dilution cultures with IL-2 (for 7-8 days). In euthymic mice, in contrast, LAK-p levels decreased for up to 6-9 days after treatment with 5-FU, Adriamycin or decarbazine, later recovering to pretreatment levels, whereas CY markedly increased LAK-p (up to 15-fold) when administered 6-12 days before limiting-dilution culture initiation. The effect of chemotherapy on LAK and NK activity was essentially similar. In other experiments, a subset of asialoGM1- LAK-p was found in the spleens of 5-FU-treated mice, but not in untreated mice. Our results suggest that the immunomodulatory effect of chemotherapy on NK/LAK activity in mice is variable and largely depends on the drug itself, the interval between chemotherapy and IL-2 administration, the strain of mice and the assay used.

Responses of human fetal splenocytes and thymocytes to interleukin-2 and comparison with adult peripheral blood lymphocytes

By means of cytotoxicity and thymidine uptake assays, the effect of human recombinant interleukin-2 (rIL-2) on the induction of lymphokine-activated killer (LAK) cell activity and cellular proliferation in splenocytes and thymocytes from human fetuses (18–22 weeks) was studied and compared with that in mononuclear cells from adult peripheral blood. It was shown that the fetal splenocytes and thymocytes incubated with low doses of rIL-2 (10–100 U/ml) developed a broad spectrum of antitumor activity (LAK cytotoxicity), although the kinetics and magnitudes of the responses were different. It was also suggested that LAK precursors were present in fetal spleen and thymus. Furthermore, rIL-2 induced a stronger proliferative and cytotoxic response in splenocytes than in thymocytes. The human fetal LAK cells isolated from the spleen were found to be 10–20 times more potent than those from adult peripheral blood with regard to cell proliferation and cytotoxicity in vitro.

IL-2, IL-3, and IFN-gamma differently affect in vivo frequencies of circulating precursors of cytotoxic T lymphocytes (CTL-P).

Experimental animal and human in vivo studies have previously demonstrated the impact of exogenous administration of various cytokines on frequencies of circulating myeloid and LAK precursor cells. For the first time we investigated whether exogenous cytokines, in the absence of antigenic challenge, may also influence frequencies of circulating antigen-specific cytotoxic T-lymphocyte precursor cells. We further asked whether triggering of autoimmune pathways as has been reported for several cytokines can be confirmed on the cellular level by demonstration of induction of autoreactive CTL-p. Limiting dilution analysis was used to determine alloreactive CTL-p frequencies in 31 patients with nonhematologic diseases before and after short-term systemic treatment with either rIL-2 (4.8 x 10(6) IU/m2 bid), rIL-3 (2.5, 5.0 or 10.0 micrograms/kg qd), rGM-CSF (5 micrograms/kg qd), rIFN-gamma (200 or 400 micrograms qd), or IFN-alpha (3 or 5 x 10(6) IU qod). Simultaneously, autoreactive CTL-p frequencies were determined by split-well analysis in 25 of these patients. We found that rIL-2 significantly expands the circulating precursor pool of alloreactive CTL (p < 0.05). rIL-3 affected CTL-p frequencies in a dose-dependent fashion. Low and intermediate doses of rIL-3 did not exhibit significant effects, whereas 10 micrograms/kg rIL-3 led to expansion of alloreactive CTL-p in the same order of magnitude as did rIL-2. This effect was statistically significant when compared with rGM-CSF (p < 0.02), which apparently had no influence on alloreactive CTL-p frequencies. In contrast to rIL-2 and rIL-3, exogenous rIFN-gamma markedly reduced the circulating precursor pool of CTL. This again was statistically significant compared with rIFN-alpha (p < 0.03), which, like rGM-CSF, did not exhibit any effects on the level of alloreactive CTL-p. Frequencies of autoreactive CTL-p were invariably below the limit of detection in our system (< 1/300,000). In conclusion, these data demonstrate that (a) short-term systemic administration of rIL-2, rIL-3, and rIFN-gamma differently affects the clone size of circulating precursors of alloreactive CTL in man, while rGM-CSF and rIFN-alpha do not exhibit measurable effects, and (b) none of the cytokines administered is capable of uncovering detectable frequencies of autoreactive CTL-p.

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

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

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

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

The Effects of Pentoxifylline on the Generation of Human Lymphokine-Activated Killer Cell Cytotoxicity

Pentoxifylline (PTX) is a methylated xanthine that has been shown to reduce the toxicity of Interleukin-2 (IL-2) therapy in animal models, possibly by inhibiting secretion of tumor necrosis factor-alpha. However, the use of PTX to reduce IL-2 toxicity in cancer patients would be advantageous only if PTX did not abrogate antitumor effector mechanisms. We therefore tested the effects of PTX on the induction of lymphokine-activated killer (LAK) cell reactivity in human peripheral blood mononuclear cells (PBMCs). LAK precursor and effector activity were defined as lysis of Daudi cells by PBMCs after incubation with IL-2 at 1,000 U/ml (LAKp) or without incubation (LAKe). PBMCs from four healthy subjects were cocultured for 5 days with IL-2 and PTX (0-1.0 mM). LAKp was inhibited in a PTX dose-dependent manner, as the mean % lysis at an E:T ratio of 50:1 was 62, 43, and 8% in the presence of 0, 0.1, and 1.0 mM PTX, respectively. To determine the effect of in vivo PTX on LAKp activity, four healthy subjects were tested after treatment with PTX at 2 g/day for 3 days. LAKp activity was detected in all four and was not inhibited by autologous serum containing PTX and biologically active metabolites. Because lymphocytes "preactivated" by IL-2 in vivo may respond to PTX differently than resting cells, two patients were tested after a 5 day infusion of IL-2 at 6 x 10(6) U/m2/day. PBMCs from both displayed LAKp activity, with far less inhibition by PTX than in resting cells (4, 17, and 37% inhibition at 0.05, 0.1, and 1.0 mM, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Lnterleukin-2 ± Lymphocytes as Consolidative Immunotherapy after Autologous Bone Marrow Transplantation for Hematologic Malignancies

Patients who undergo autologous bone marrow transplantation (ABMT) for advanced hematologic malignancies experience a high relapse rate. Therapy with interleukin-2 (IL-2) +/- lymphokine-activated killer (LAK) cells has induced clinical responses in some patients with advanced malignant lymphoma (ML) or acute myelogenous leukemia (AML). It is postulated that IL-2 +/- LAK cells represents a potentially non-cross-resistant therapeutic modality which might prevent or delay relapses if used as consolidative immunotherapy after ABMT, at a time of minimal residual disease. Therefore, we first studied the reconstitution of IL-2-responsive LAK precursor cells after ABMT and found them in the circulation as early as 3 weeks after ABMT. A phase Ib clinical trial was then performed which identified a tolerable IL-2 regimen which could be administered early after ABMT and which could induce immunomodulatory effects. We then initiated a clinical trial to determine the feasibility of generating and administering autologous LAK cells using this IL-2 regimen after ABMT for 16 patients with ML. The results show that IL-2+LAK therapy early after ABMT is feasible but is more toxic than IL-2 alone. Patients with AML on the phase I IL-2 trial and with ML on the IL-2+LAK protocol were evaluated for tumor status. Of 8 patients with AML in first relapse or at a later stage who underwent ABMT and received IL-2, 2 have relapsed, while 6 remain in complete remission 26+ to 40+ (median 28+) months after ABMT. Of 16 patients with ML considered at high risk for relapse who were treated with ABMT+IL-2+LAK, 5 have relapsed, while 11 remain in complete remission at 6+ to 21+ (median 10+) months after ABMT. The results in both trials are quite encouraging and appear to be better than those in nonrandomized historical controls at our institution. Prospectively randomized trials of IL-2 versus no IL-2 after ABMT in such patients are being initiated to assess definitively the effect, if any, on the relapse rate.

Study of the effect of lithium on lymphokine-activated killer cell activity and its antitumor growth.

The in vitro effect of lithium on lymphokine-activated killer cell (LAK) activity and its in vivo antitumor growth were observed. LAK activity was enhanced when LiCl was added during LAK cell induction, and this enhancement was observed both in human peripheral blood mononuclear cell and in mouse splenocytes used as LAK precursors. Cholera toxin, which can increase intracellular levels of cAMP, decreased LAK cell activity. However, lithium partially reversed this inhibitory effect, indicating that lithium increased LAK cell activity by decreasing cAMP levels. D-Sphingosine, an inhibitor of protein kinase C, and EGTA, a calcium chelator, both inhibited the LAK cell activity. However, their inhibitory effects could not be reversed by lithium because lithium was added in the culture in combination with one of these inhibitors during LAK cell induction. By using slot blot analysis, the effect of lithium on the expression of tumor necrosis factor-alpha mRNA of LAK cells was analyzed. Lithium increased the level of tumor necrosis factor-alpha mRNA when both lithium and interleukin 2 were added to induce LAK cells. The in vivo antitumor effect of lithium has also been studied. Using a mouse melanoma experimental model, the effect of lithium on tumor growth was also observed. Both lithium alone and interleukin 2/LAK had an antitumor effect, whereas the treatment of interleukin 2/LAK in combination with lithium had the strongest inhibitory effect on tumor growth, since this treatment resulted in reduction of tumor size and prolongation of survival in tumor-bearing mice. Therefore, it is hopeful that lithium can be used as a new immunomodulator for cancer immunotherapy and immune diseases.

Human lymphokine-activated killer cells are cytotoxic against cells infected with Toxoplasma gondii.

Experiments were conducted to determine whether human lymphokine-activated killer (LAK) cells are cytotoxic against cells infected with Toxoplasma gondii. Nylon wool nonadherent (NWNA) peripheral blood lymphocytes, as well as purified natural killer cell (NK) (CD3- CD16+ CD56+) and T (CD3+ CD16- CD56-) cells obtained from five healthy T. gondii seronegative volunteers exhibited minimal cytotoxic activity against T. gondii-infected cells. When standard LAK (S-LAK) cell preparations were induced by incubation of NWNA cells with recombinant interleukin 2, induction of remarkable cytotoxic activity against T. gondii-infected cells. When standard in LAK cell preparations from each of the volunteers. The phenotype of the LAK precursor and effector cells varied depending on the target cell used. Whereas the precursor and the effector cells of most of the LAK activity against K562 and Daudi cells were cells with NK phenotype, when T. gondii-infected cells were used as targets, both cells with NK and T cell phenotypes were precursors and effectors of the lysis. When cytotoxic activity of S-LAK cells was compared with the activity of adherent LAK (A-LAK) cells, A-LAK cells displayed higher cytotoxic activity against T. gondii-infected cells, as well as against K562 and Daudi cells. Cold target inhibition experiments suggested that there is a subset of LAK effector cells capable of lysing both T. gondii-infected cells and Daudi cells, whereas other subsets preferentially or exclusively lyse one of these target cells.

The distribution of the CDW52 molecule on blood cells and characterization of its involvement in T cell activation.

The O97 mouse mAb was used to define, together with the CAMPATH-1 mAb series, the CDw52 in the course of the Fourth International Workshop on Human Leucocyte Differentiation Antigens. O97 and CAMPATH-1M produce full competitive inhibition and react with an epitope dependent on O-linked sugar residues. The two mAb, however, display significant differences in reactivity with leukocyte populations--in fact the reactivity of O97, which also exerts a powerful cytotoxic effect with human C', is similar to mAb from the CAM-PATH-1 series having the broadest one. Noteworthy, O97 spares PBL, NK, and LAK precursors, permitting an easy purification of these cells; O97 is able to kill long-term colony-forming cells in bone marrow culture and characteristically reacts, in contrast to CAMPATH-1M, with erythrocytes. Western blotting has revealed a strikingly different molecular size on red cells, since CDw52 mAb revealed a broad band ranging from 6-16 kDa, instead of the 21-28 kDa revealed from leukocyte extracts. In agreement with immunofluorescence data, O97 revealed abundant material from red cells in Western blotting, while reactivity of CAMPATH-1M was very faint. Overall, these results show that distinct molecular forms of the CDw52 molecules are present on different blood cells. We have also characterized an activation signal that can be delivered to T cells via the CDw52 molecule by CAMPATH-1M but not by O97. This is an accessory signal that can complement a primary activation signal delivered via the CD2 pathway but not via the CD3-TcR pathway. This is similar to the effects obtained with the CD45R (2H4) mAb, 2H4 and CAMPATH-1M mAb having additive effects on T cell activation via CD2.

Effective in 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 observed in vivo. Total number of splenocytes and the ratio of asGM1+ cells increased gradually after i.v. administration of OK-432, reaching their peaks at 3 to 4 days. It was found that asGM1+ 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 x 10(4) 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 asGM1 antibody prior to rIL-2 injection abolished completely such augmentation of LAK activity in OK-432 treated mice. These results demonstrated that asGM1+ LAK precursor cells induced by OK-432 were effectively differentiated into LAK cells by rIL-2.

The mechanism of IL-2-mediated protection against GVHD in mice. II. Protection occurs independently of NK/LAK cells.

We have recently demonstrated that high-dose IL-2, when begun on the day of bone marrow transplantation, has a potent protective effect against graft-vs.-host disease mortality, especially when coadministered with T cell-depleted syngeneic bone marrow cells. Because several groups of investigators have demonstrated that lymphokine-activated killer cells can mediate GVHD protection, we hypothesized that the mechanism of protection by IL-2 administration might involve the in vivo activation of natural killer and/or LAK cells. In order to test this hypothesis, we evaluated the effect of IL-2 administration on the number of NK1+ cells and on NK-mediated cytotoxic activity in recipients of GVHD-producing inocula. Furthermore, we evaluated the effects on IL-2-induced GVHD protection of depleting NK cells and LAK precursor cells in vivo with mAb against NK1.1 or antiserum against asialo GM1. The results demonstrate that: (1) The number of NK1+ cells is not increased in spleens of IL-2-treated compared with control recipients of GVHD-producing inocula; (2) NK activity is not increased in IL-2-treated compared with control recipients of GVHD-producing inocula during or immediately following the period of IL-2 administration; (3) depletion of NK cells and LAK precursors from the donor and host influenced the time course of GVHD-related mortality in a complex fashion; and (4) IL-2-induced GVHD protection is largely independent of the activity of an NK or LAK cell population of donor or host origin. IL-2-induced GVHD protection therefore reflects primarily the activity of non-LAK protective cell populations, or it may be a direct inhibitory effect on responding donor cell populations as they encounter host antigen.

Intestinal lymphokine‐activated killer cells in inflammatory bowel disease

The role of non-specific cytotoxicity in the pathogenesis of inflammatory bowel disease (IBD) was investigated by assaying the natural killer (NK) and lymphokine-activated killer (LAK) cell activity of lamina propria mononuclear cells (LPMC) from 22 specimens of intestinal mucosa affected by IBD. Only minimal levels of NK activity were detected against K562 cells, as well as colon carcinoma cells, adenoma cells and fibroblasts freshly isolated from the intestinal mucosa. Culture of LPMC from IBD in the presence of interleukin-2 (IL-2) generated LAK cells that mediated high levels of activity against K562 cells and against neoplastic epithelial cells and fibroblasts derived from the intestinal mucosa. A group of 20 histologically normal specimens of intestinal mucosa showed similar levels of LAK activity against the K562 and intestinal cell targets. The minimal mucosal NK activity in IBD suggests that the cytotoxic properties of NK cells are not important in the pathogenesis of IBD. The presence of LAK precursor cells in the inflamed mucosa of IBD and their ability to lyse biologically relevant targets in vitro suggests that LAK cells have the potential to contribute to intestinal mucosal injury in IBD.

Toxicity and immunomodulatory effects of interleukin-2 after autologous bone marrow transplantation for hematologic malignancies

Autologous bone marrow transplantation (ABMT) for advanced hematologic malignancies is associated with high relapse rates. Interleukin-2 (IL- 2) and lymphokine-activated killer (LAK) cells represent a potentially non-cross-resistant therapeutic modality that might prevent or delay relapses if used early after ABMT at a time when the tumor burden is minimal. However, high-dose chemoradiotherapy and ABMT might increase patients' susceptibility to IL-2 toxicity, and might interfere with immunologic responses to IL-2 in vivo. Therefore, to determine safety, tolerance, and immunomodulatory effects of IL-2 therapy early after ABMT, IL-2 was administered by continuous intravenous infusion to 16 patients 14 to 91 days (median, 33) after ABMT for acute leukemia, lymphoma, or multiple myeloma. Patients were sequentially assigned to escalating IL-2 “induction” doses (0.3 to 4.5 x 10(6) U/m2/d, days 1 to 5), and all patients received a nonescalating IL-2 “maintenance” dose (0.3 x 10(6) U/m2/d, days 12 to 21). Most patients exhibited mild to moderate fever, nausea, diarrhea, and/or skin rash with IL-2 infusions. The maximum tolerated “induction” dose was 3.0 x 10(6) U/m2/d; dose- limiting toxicities were hypotension and thrombocytopenia. All toxicities reversed on stopping the IL-2 infusions, and all patients completed “maintenance.” Postinfusion lymphocytosis was exhibited by patients at all IL-2 dose levels. With the higher IL-2 doses, increased percentages of patients' PBMC expressed CD16 and CD56, with augmented lysis of K562 and Daudi, reflecting the induction of natural killer and circulating LAK effector activities. Increased LAK precursor activity was exhibited by patients at all IL-2 dose levels. Thus, the IL-2 therapy regimen was safely tolerated after ABMT, and pronounced immunomodulatory effects were observed with the higher IL-2 doses. These studies support the planned use of IL-2 and LAK cells after ABMT in an attempt to reduce relapses of advanced hematologic malignancies.