ANTI-HUMAN LUNG GIANT CELL CANCER (PG) EFFECT OF HUMAN LAK CELLS IN VITRO AND IN NUDE MICE

Human LAK cells were prepared by culturing normal human peripheral blood mononuclear cells (PBMC) with or without rIL-2 and assayed for T cell surface markers as well as anti-tumor activity against PC in vitro and in nude mice. Although the percentages of T3, T4, and T8 positive cells in rIL-2-activated cells did not differ significantly from those of control cells in vitro, the former showed stronger cytotoxicity than control cells to PG tumor cells in vitro. In vivo, LAK cells completely inhibited the growth of PG tumor in nude mice, whereas PBMC control cells were to be of no effect. The anti-tumor effect of human LAK cells in nude mice may offer a useful model to study the role of human LAK cells against human tumor in vivo.

THE EFFECT OF THE TRANSFER OF LAK CELLS, COMBINED WITH ALLOIMMUNIZATION, ON THE PULMONARY METASTASES OF RAT MAMMARY CARCINOMA

Alloimmunization was combined with lympho-kine activated killer (LAK) cells to assess its effect on mammary carcinoma in rats. The animals were injected with both irradiated allosplenocytes and syngeneic LAK cells. Metastatic lung nodules were markedly reduced using combined therapy when compared with the transfer of LAK cells or alloimmuni-zation alone. IL-2 activity in the serum of alloim-munized rats could be detected. This activity, maintained in vivo for one week, may be responsible for enhancing the antitumor effect of transferred LAK cells.

TREATMENT OF PATIENTS WITH MALIGNANT PLEURAL EFFUSIONS DUE TO ADVANCED LUNG CANCER BY TRANSFER OF AUTOLOGOUS LAK CELLS COMBINED WITH rIL-2 OR rIL-2 ALONE

Experimental study both in vitro and in vivotogether with clinical trials showed that LAKcells have antitumor and antimetastatic effects(1-5)and that these effects are closely related tothe number of LAK cells transferred and the ad-ministration of rIL-2(1,6-8).Usually,autologousPBL’s are used as the source of LAK precursorsin the adoptive immunotherapy of cancer patients.But this not only puts an added burden on thecancer patient,it can cause serious side effectsas well(9).Although TIL’s may provide a solu-tion to this problem(10,11),their isolation fromsolid tumors is complex and consumes many rea-gents.We have reported that the isolation oflymphocytes from malignant ascites or from ma-lignant pleural effusions is not only simple

A STUDY OF THE CYTOTOXICITY OF MALIGNANT PLEURAL EFFUSION LYMPHOCYTES AND LAK CELLS AGAINST AUTOLOGOUS TUMOR CELLS

The cytotoxicity of malignant pleural effusion lymphocytes (MPEL) against autologous tumor cells (ATC) were compared with that of peripheral blood lymphocytes (PBL). It was demonstracted that the cytotoxicity of PBL was higher than that of MPEL (P< 0.001), but the cytotoxicity and expansion of MPEL-activated by rIL-2 was much higher than that of PBL activated by rIL-2 (LAK cells) (P< 0.001). This shows that local immune reaction of the pleural cavity of patients with malignant pleural effusion was in the state of suppression. MPEL activated are better effector cells than LAK cells in tumor adoptive immunotherapy.

Study of Multi-directional Derivation of Cord Blood Mononuclear Cells and Observation of Killing Activity to MGC-803 Gastric Cancer Cell Strain In Vitro

Objective： To study multi-directional derivation of cord blood mononuclear cells to CD3AK, LAK and CIK cells as well as changes of killing activity to gastric cancer cell strain in vitro. Methods： CD3mAb and IL-2 were used to induce CD3AK cells, and IL-2 was used to induce LAK cells; IFN-γ was used in the beginning, then IL-1, CD3mAb and IL-2 were used to induce CIK cells after 24 h for observing amplification and analyzing their relationship. The phenotypes of the cultured CIK cells were analyzed by flow cytometry. Subsequently, by using MGC-803 gastric cancer cell strain as target cells, the killing activity of CD3AK, LAK and CIK cells was evaluated by using MTT method. Results： The amplification activity of CD3AK and CIK cells was all far higher than LAK cells （P〈0.05）. The amplification activity had no obvious difference between CIK cells and CD3AK cells at prophase, but that was far higher in CIK cells than CD3AK cells at about 20^th day （P〈0.05）. The flow cytometry revealed that the amount of CD3^＋ CD56^＋ cells, major effector cells after CIK cells being cultured was significantly increased （P〈0.05）, moreover, the amount of CD8^＋ cells was significantly increased as well （P〈0.05）. The killing activities of CD3AK and CIK cells to the MGC-803 gastric cancer cell strain were all significantly higher than LAK cells, while the killing activity of CIK cells was stronger than CD3AK cells （P〈0.05）. Conclusion： CIK cells have stronger amplification activity and killing activity, and can be taken as more effective killing cells applied to the tumor adoptive immunotherapy.

APOPTOSIS OF TUMOR CELLS IN LECTIN－DEPENDENTLYMPHOKINE－ACTIVATED KILLER CELLMEDIATED CYTOTOXICITY

By using DNA electrophoresis and propidium iodide(PI) staining flow cytometry(FACS) analysis, we studied the mechanisms of lymphokine-activated killer(LAK) cell-mediated cytotoxicity. In the presence of pokeweed mitogen(PWM), human LAK cells induced DNA fragmentation of two leukemic cell lines(U937 cells and Raji cells) and two solid tumor cell lines(SW1116 cells and Hep-2 cells), a hallmark of apoptosis. The reactions were carried out at the effector/target ratio of 1:1 in 4h co-culture. Pretreatment with RNA and protein synthesis inhibitors(actinomycin D and cyclo hexamide) did not prevent the target cells from apoptosis.As the TNF-resistant tumor cell lines such as SW1116 cells and Raji cells were also triggered to apoptosis, other factors than TNF may play the role. DNA-PI staining FACS analysis also suggested that a part of LAK cells underwent apoptosis to some extent during incubation with target cells. The results provide a new way to investigate the mechanisms of the cytotoxicity of LAK cells, and a new possibility to enhance the efficiency of adoptive tumor therapy with LAK cells.

THE IN VITRO POTENTIATION OF LAK CELL CYTOTOXICITY IN CANCER AND AIDS PATIENTS INDUCED BY F3 A FRACTIONATED EXTRACT OF ASTRAGALUS MEMBRANACEUS

The in vitro induction of LAK cell activity was studied in cancer and AIDS patients. F3, an immuno regulatory component of Astragalus membranaceus was shown capable of potentiating LAK cell activity induced by rIL-2. The LAK cells killing activity against Hs294T melanoma cell line induced by 50 U/ml rIL-2 in the Presence of F3 (55 μg/ml) reached 64%, which was comparablc to that (60%) induced by 500 U/ml of rIL-2alone. With F3 and rIL-2, the effcctor to target ratio could be reduced to one-half in order to obtain an equivalent level of cytotoxicity induced by rIL-2 alone.In some patients whose Peripheral blood Iymphocytes were relatively inert of rIL-2, F3 could make them responsive to rIL-2 induction. These results imply that F3 may be useful to potentiate LAk cell activity, reduce the dosage of rIL-2 and thus minimize the later's toxic side effects when used in vivo.

Experimental Studyon Anti－tumor Effect of SplenocytesInduced by Anti－CD3McAb，PHA and IL－2

The proliferation of splenocytes from healthy adults was induced by anti-CD3 McAb,PHA and IL-2.The proliferative capability and anti-tumor activity as well as phenotypes of the splenocytes cultured in different medium systems were studied.The results showed that anti-CD3 McAb and PHA not only enhanced the proliferation of splenocytes induced by IL-2,but also produced synergism if used simultaneously.The expressions of CD4 and Tac of cellular surface markers were increased after splenocytes were induced by anti-CD3 McAb and PHA.The results of anti-tumor activity of LAK cells suggested that PHA had the capability to promote anti-tumor activity of LAK cells by both direct and in direct pathways,but anti-CD3 McAb indirectly promoted anti-tumor activity of LAK cellsby enhanctng splenocyte proliferation.

Immunomodulatory Effects of Esculetin(6,7-Dihydroxycoumarin)on Murine Lymphocytes and Peritoneal Macrophages

Coumarins belong to a diverse group of naturally occurring non-nutrient phytochemicals known as benzo-α- pyrones. In this study, esculetin, a 6,7-dihydroxy derivative of coumarin with pleiotropic biological activities, was found to have no significant cytotoxic effect on normal murine macrophages, but it could increase the in vivo migration of the thioglycollate-elicited macrophages in a dose-dependent manner. Moreover, esculetin significantly increased the endocytic activity, and augmented the nitric oxide production and iNOS gene expression in LPS-treated macrophages. In addition, in vivo administration of esculetin into mice was shown to increase the mitogenesis of splenic lymphocytes towards Con A and LPS stimulations, and induced the LAK activity of splenic lymphocytes. Collectively, our results indicate that esculetin could exert immunomodulatory effects on murine macrophages and lymphocytes, both in vitro and in vivo, and this might be one of the possible mechanisms by which coumarins can exert their chemopreventive and anti-tumor activities in vivo. Cellular ＆ Molecular Immunology. 2005;2（3）： 181-188.