The anti-tumor efficacy of lymphokine-activated killer (LAK) cells induced in vitro from peripheral blood lymphocytes of patients with malignant glioma

We studied whether lymphokine-activated killer (LAK) cells were capable of being induced in vitro from peripheral blood lymphocytes (PBL) of patients with malignant glioma, by using recombinant IL-2 (rIL-2). We then investigated whether they possessed anti-tumor efficacy against malignant gliomas (ONS-12, -20, -44). Human LAK cells were generated by placing 5 X 10(6) PBL into each well of 24-well plates (Corning) containing 2 ml of complete medium (CM) with 10 units of rIL-2 (TGP-3, provided by TAKEDA Chemical Industries, Ltd.). The CM consisted of RPMI 1640 with 0.1 mM nonessential amino acids, 1 microM sodium pyruvate, 5 X 10(-5) M 2-mercaptoethanol, 50 micrograms/ml gentamicin sulfate, 0.03% glutamine and 1% heat-inactivated human AB serum. The plates were incubated horizontally at 37 degrees C in a 5% CO2 atmosphere for 72-96 hours. The LAK cells were then harvested, washed three times with Hanks balanced solution, and resuspended in RPMI 1640 with 1% heat-inactivated human AB serum for the in vitro cytotoxicity assays. The anti-tumor cytotoxic activity of LAK cells was estimated in triplicate by 4-hr 51Cr release assays. The cytotoxic activity of the LAK cells against autogeneic ONS-44 glioma cells and PHA blasts was approximately 30% and a few %, respectively. The Natural Killer (NK) activity of the patient with ONS-44 glioma cells was equivalent to that of healthy subject.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clinical studies of adoptive immunotherapy of human disseminated brain tumors with LAK cells and recombinant interleukin-2

In previous in vitro studies, the authors showed that recombinant interleukin-2 (rIL-2) stimulated peripheral blood lymphocytes (PBL) from patients with malignant brain tumors to generate cells that were lytic for fresh autologous tumor but not for lymphocytes or lymphoblasts. We then tried the adoptive transfer of such lymphokine-activated killer-(LAK) cells induced from patients with meningeal gliomatosis (MG) and meningeal carcinomatosis (MC). PBL fractions were separated into a plastic bag by leukophoreses using HEMONETICS V 50. PBL were then harvested by LSM (Litton Bionetics, Kensington, Md.) gradient centrifugation according to the standard method. Human LAK cells were generated by placing 5 X 10(7) PBL in 10-cm diameter plastic dishes (Falcon) holding 20 ml of complete medium (CM) containing 100 units of rIL-2 (TGP-3, provided by TAKEDA Chemical Industries, Ltd.). The CM consisted of RPMI P640 with 0.1 mM nonessential amino acids, 1 microM sodium pyruvate, 5 X 10(-5) M 2-mercaptoethanol, 50 micrograms/ml of gentamicin sulfate, 0.03% glutamine and 2% heat-inactivated human AB serum. The dishes were incubated horizontally at 37 degrees C in a 5% CO2 atmosphere for 72-96 hours. The LAK cells were then harvested, washed three times with Hanks' balanced salt solution (HBSS) and resuspended in HBSS for transfer to patients. 1-2 X 10(8) LAK cells were injected intrathecally through Ommaya's reservoir or a V-P shunt reservoir twice a week. Portions of LAK cells were tested for cytotoxicity in vitro. During the adoptive transfer of LAK cells, patients were given intravenous injections of 500 units of rIL-2 every day. Case 1: A 29-year-old man with meningeal gliomatosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of phenytoin on cell-mediated immunity

Phenytoin is a highly effective anticonvulsant agent that is widely administrated to prevent some kinds of patients with brain tumor. But it has been said that phenytoin may have some immunosuppresive potential for hosts. In this study, we evaluated the effects of phenytoin upon cellular immunity such as NK, CTL and LAK activity in murine models. Fresh splenocytes were taken out from mice (CBA/J, C 3 H/HeN, C 57 BL/6) into which phenytoin had been injected intraperitoneally at a daily dose of 1,000 micrograms for 28 days. The serum concentration of phenytoin in the experimental models was 10-20 micrograms/ml. The cytotoxic activities were estimated by a 4-hr 51Cr release assay. The mitogen-stimulated lymphocyte function was evaluated by 3H-thymidine incorporation into DNA. The NK activity was estimated by cytotoxicity of splenocytes of CBA/J mice against NK-sensitive YAC-1 cells. The cytotoxic T-lymphocyte (CTL) activity was estimated by cytotoxicity of splenocytes of C 57 BL/6 mice which were stimulated in vitro for 5 days by splenocytes of C 3H/HeN treated with mitomycin C, against RSV-M glioma cells. Lymphokine-activated killer (LAK) activity was estimated by cytotoxicity of LAK cells, which were induced from splenocytes of C 3 H/HeN mice by human recombinant interleukin-2 (rIL-2), against syngeneic RSV glioma and allogeneic 203 glioma cells. 3H-thymidine incorporation of splenocytes of C 57 BL/6 mice was reduced significantly (p less than 0.01) in phenytoin-treated mice. The cytotoxicity of splenocytes of non-treated CBA/J mice against YAC-1 cells was 75%, but that of phenytoin-treated CBL/J mice was a few %.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of anticonvulsants on cellular immunity

The effects of anticonvulsants on cellular immunity were examined in murine models. Fresh splenocytes were obtained from mice which had been intraperitoneally given 1 mg of phenytoin, 2 mg of phenobarbital, or 20mg of valproate for 28 days. The serum concentration of phenytoin, phenobarbital and valproate in these animals were 10-20 micrograms/ml, 30-40 micrograms/ml and 50-70 micrograms/ml, respectively. The proliferative response of splenocytes to mitogens was assessed by 3H-thymidine incorporation. The cytotoxic activities of cells such as natural killer (NK) cells, cytotoxic T lymphocytes (CTL), and lymphokine-activated killer (LAK) cells were estimated by a 4 hr-51Cr release assay. Phenytoin suppressed lymphocyte proliferation, NK activity, and CTL activity, but never LAK activity. Phenobarbital suppressed proliferative response to rIL-2 and CTL activity, but did not suppress NK activity nor LAK activity. In turn sodium pyruvate never suppressed any activity on cellular immunity.     

Adoptive immunotherapy in experimental brain tumor in rats--induction of LAK cells and their biological characteristic

We have investigated the biological characteristic of Lymphokine Activated Killer (LAK) cells induced from spleen cells (SPC) of Fischer rats. Supernatant of 48 hour culture medium (RPMI-1640, 10% FBS, 2-ME: 5 X 10(-5) M, HEPES: 10 mM) of SPC (1 X 10(6)/ml) from Wistar rats in the presence of Con A (5 micrograms/ml) was used as Interleukin 2 (IL-2). LAK cells were generated by co-cultivation of SPC (4 X 10(6)/ml) from Fischer rats with peak reactivity on the 2 nd or 3 rd day of culture. Lytic activity was observed against not only syngeneic tumor cells (T9, RSV induced brain tumor), but also allogenic (KMT-17) and xenogenic (K 562, Raji cell, G 361, YAC-1) tumor cells, while no lytic activity was observed against normal brain cells. Cell depletion test, dye exclution test and immunofluorescence method using monoclonal Antibodies (mAbs) revealed that LAK cells partially belonged to the population of activated T cell group (W 3/13, OX 4 positive) but the precursor cells did not react with any mAbs used (W 3/13, W 3/25, OX 4, OX 8). On the basis of these results, more effective and useful administration of LAK cells is now under investigations by using the rats with brain tumors.     

Establishment and biological characterization of human medulloblastoma cell lines

Two cell lines of human medulloblastoma (ONS-76 and ONS-81) were established, and their biological characteristics were investigated. The cell line, ONS-76, was established from a tumor specimens obtained from a large cerebellar tumor of a 2-year-old girl. The pathological diagnosis was a typical medulloblastoma. The other cell line, ONS-81, was derived from a metastatic tumor in right frontal lobe of a 9-year-old girl. The tumor specimens were minced into fragments approximately 1 mm in diameter and cultured in plastic culture flasks in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS) and 50% patients serum. The cells growing as a monolayer were subcultured in RPMI 1640 supplemented with 10% FCS and initially with L-glutamine, sodium pyruvate, and nonessential amino acid. Microscopically, both cultured cells exhibited various morphological appearances, and this morphological heterogeneity seemed to be specific for medulloblastoma cells. The in vitro population doubling time of ONS-76 and ONS-81 were 18.6 and 19.2 hr, respectively. The ONS-76 and ONS-81 cells formed subcutaneous tumors in nude mice as serial transplantable xenograft, and these tumors had a microscopic appearance similar to that of the original medulloblastoma. Ultrastructurally++, the cultured cells showed primitive, undifferentiated appearance, and no neuronal or glial structures were not seen. Immunohistochemical studies showed that both cells expressed neuron-specific enolase (NSE) and neurofilament protein (NFP 200 K, 145 K), but glial fibrillary acidic protein (GFAP) and S-100 protein were not detected. The NFP immunoreactivities of both cultured cells were demonstrated as abnormal perinuclear deposits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Observations on the local administration of autologous lymphokine activated killer cells and recombinant interleukin-2 in patients with malignant gliomas

Recently lymphocytes from patients with cancer have proved to be activated by interleukin 2 (IL-2), and show a strong cytotoxicity. On the basis of this fact, we have tried to inject lymphokine activated killer (LAK) cells and recombinant IL-2 (rIL-2) directly into the cavity of brain tumor. We describe here preliminary results of the local administration of LAK cells and the rIL-2 to patients with malignant gliomas. Lymphocytes from the patients were separated from venous blood on a Ficoll gradient. By culture with rIL-2 for five days, the lymphocytes were activated to generate LAK activity, which was measured by chromium release assay. These LAK cells were capable of killing various kinds of tumor cells including their own cells. For example, their LAK activity to Daudi cell and self tumor cells was approximately 66 and 49%, respectively. These LAK cells showed a strong killing activity in excess of 40 to 70% against various tumor cells. Furthermore, activated killer cells, such as LAK cells, phytohemagglutinin-activated killer cells, and their precursor cells were serologically studied for the recognition of their biological characteristics. The phenotype of these LAK cells were sensitive to Leu 1, 3a, 7, and extremely so to 11 monoclonal antibodies, whereas LAK precursors were mainly sensitive to Leu 11 monoclonal antibodies. This observation led us to think that LAK cells belonged to the polyclonal cell populations. Following the fundamental studies, we applied this adoptive immunotherapy to 12 patients with malignant gliomas in whom standard therapy turned to be unsuccessful. All patients had histological evidence and progressive disease in spite of standard radiochemotherapy and other treatments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lymphokine activateed killer (LAK) cell-mediated lysis of murine glioma: trypsinchymotrypsin-sensitive glioma protein is responsible for tumor-selective recognition by LAK cells

The killing of Fischer rat 9L glioma in vitro by lymphokine-activated killer (LAK) cells was studied. LAK cells generated by culturing Fischer spleen cells with recombinant interleukin 2 markedly lysed glioma cells but did not kill syngeneic normal brain tissue in a chromium release microcytotoxicity assay. Susceptibility of glioma to lysis by LAK cells was markedly diminished by pretreating the glioma cells with trypsin or chymotrypsin but was unaffected by pretreatment with neuraminidase, glycosidases, or sodium periodate. These results suggest that LAK cell killing of glioma is probably tumor-selective and that a crucial cell surface determinant on glioma cells responsible for its tumot-selective lysis by LAK is a protein sensitive to trypsin and chymotrypsin.     

Recombinant human prolactin improves antitumor effects of murine natural killer cells in vitro and in vivo

OBJECTIVE: To verify the effect of prolactin on natural killer (NK) cells in vivo and its implications for NK cell immunotherapy. METHODS: Recombinant human prolactin (rhPRL; 30 microg, i.p.) was administered to BALB/c mice and severe combined immunodeficiency (SCID) mice 1 day before harvesting splenocytes for (51)Cr release assay to examine the effects of rhPRL on NK cells of normal mice. rhPRL (10 microg, i.p., every other day for a total of 5 injections) was administered to BALB/c mice after syngeneic bone marrow transplantation (SBMT) to determine its effects on NK cell reconstitution. CT26 tumor cells were injected into BALB/c mice intravenously on day 0, and interleukin (IL)-15-cocultured activated syngeneic NK cells (IL-15-NK) from SCID mice were intravenously injected into tumor-bearing BALB/c mice on day 1. The improvement of antimetastasis effects and survival of tumor-bearing mice by rhPRL were checked. RESULTS: BALB/c and SCID mice receiving one rhPRL injection exhibited a significant increase in cellular cytotoxicity against YAC-1 target tumor cells; the specific lysis was enhanced from 12.5 to 17.3% in BALB/c mice and from 27.8 to 51.2% in SCID mice. BALB/c mice continuously receiving rhPRL injections exhibited significant increases in NK cell (DX5-positive) contents and cellularity in both the bone marrow and spleen in the SBMT model. The bone marrow NK cell contents were improved from 1.53 to 3.13% after rhPRL injection. NK cells from SCID mice were then cultured with recombinant human IL-15 (rhIL-15; 6000 IU/ml), rhPRL (10 ng/ml) or rhIL-15 plus rhPRL for 25 days. The cytotoxicity and cellularity were enhanced by rhPRL when tested on day 10, when comparing the rhIL-15 plus rhPRL group with the rhIL-15 group or rhPRL group, respectively. In the adoptive cellular immunotherapy study, the IL-15-NK plus IL-15 plus rhPRL group showed significantly lower numbers of lung metastases and longer survival than the IL-15-NK plus IL-15 group; the mean survival interval was prolonged from 31.5 to 53.7 days. CONCLUSION: These results indicate that rhPRL is possibly an important regulator of NK cells and a potential biologic product for immunotherapy.     

Interleukin-2 and lymphokine activated killer (LAK) cells in the treatment of malignant glioma: clinical and experimental studies

The phenomenon of glioma killing by lymphokine activated killer cells (LAK) was studied. We demonstrate that LAK cells generated by culturing the lymphokine interleukin-2 (IL-2) with peripheral blood lymphocytes from brain tumour patients destroys autologous glioma. The rat 9L glioma model was used to show that LAK killing was tumour-selective as glioma but not syngeneic normal brain tissue was destroyed. The susceptibility of both human and 9L rat glioma to LAK cell killing was markedly diminished by pretreating glioma cells with trypsin or chymotrypsin, but was unaffected by pretreatment with neuraminidase, glycosidases, sodium periodate or hydrocortisone. These results suggest that the cell surface determinant on glioma cells responsible for its tumour selective lysis by LAK is a protein sensitive to trypsin and chymotrypsin. The tumour-selective killing of glioma by LAK in vitro prompted the initiation of a Phase I study in which ten patients with malignant glioma have been treated with direct intracerebral injection of IL-2 or LAK without evidence of systemic or brain toxicity.     

Interleukin-2 and lymphokine activated killer (LAK) cells in the treatment of malignant glioma: clinical and experimental studies

The phenomenon of glioma killing by lymphokine activated killer cells (LAK) was studied. We demonstrate that LAK cells generated by culturing the lymphokine interleukin-2 (IL-2) with peripheral blood lymphocytes from brain tumour patients destroys autologous glioma. The rat 9L glioma model was used to show that LAK killing was tumour-selective as glioma but not syngeneic normal brain tissue was destroyed. The susceptibility of both human and 9L rat glioma to LAK cell killing was markedly diminished by pretreating glioma cells with trypsin or chymotrypsin, but was unaffected by pretreatment with neuraminidase, glycosidases, sodium periodate or hydrocortisone. These results suggest that the cell surface determinant on glioma cells responsible for its tumour selective lysis by LAK is a protein sensitive to trypsin and chymotrypsin.

The tumour-selective killing of glioma by LAK in vitro prompted the initiation of a Phase I study in which ten patients with malignant glioma have been treated with direct intracerebral injection of IL-2 or LAK without evidence of systemic or brain toxicity.     

“自体血清＋鸡尾酒法”诱导外周血DC成熟的研究

目的比较三种不同的分离诱导外周血树突状细胞（DC）成熟的方法。方法取正常成人外周血约180ml，分成三组。分离单核细胞后，经典脂多糖（LPS）诱导组（A组）：接种于含RPM11640＋胎牛血清完全培养基的25cm^2培养瓶中，2h后弃悬浮细胞，将贴壁单核细胞，用粒-巨噬细胞集落刺激因子和白介素4培养，培养到第6天时加入LPS诱导24h；鸡尾酒诱导组（B组）：单核细胞的培养同A组，培养到第6天时用细胞因子组合（1000U／ml肿瘤坏死因子、10ng／ml白介素6、10ng／ml白介素1β、1μg/ml前列腺素E2）诱导24h；自体血清＋鸡尾酒诱导组（C组）：单核细胞用RPM11640＋10％自体血清完全培养基培养，诱导方法同B组。用流式细胞仪检测各组收集的DC细胞成熟表型CD80、CD86、CD83以及白细胞表面抗原HLA-DR的表达；动态观察各组细胞成熟过程中形态的改变以及其激活淋巴细胞增殖的能力。结果C组DC成熟过程中形态好、突起多；其成熟表型CD86、CD83、CD80以及HLA-DR的平均表达率分别是（88．60±6．34）％、（50．55±4．03）％、（73．89±6．15）％、（93．24±7．78）％，明显高于A、B组（P〈0．05）；激活淋巴细胞增殖指数为2．03，亦明显高于A、B组（P〈0．05）。结论自体血清＋鸡尾酒诱导法是一种简单、方便、经济的外周血DC分离诱导成熟的新方法。     

An immune cell population that responds to beta-endorphin and is responsible for protecting nude mice from the fatal consequences of a virus infection of the central nervous system.

Reconstitution of 3- to 4-week-old BALB/c nude (nu/nu) mice with 10(7) syngeneic splenocytes, 48 h before intracerebral inoculation with a temperature-sensitive (ts) mutant of VSV (tsG31 KS5), provided protection from the fatal consequences of clinical disease in 80-90% of the infected animals. Reconstitution of animals with 10(7) splenocytes, first depleted of natural killer (NK) cells with anti-asialo GM1 and complement, also afforded protection against the infectious disease. Depletion of T-lymphocytes with anti-thy-1.2 antibody and complement, however, provided little protection with approximately 40% of the animals succumbing to the virus infection within 30 days post-infection. A single intracerebroventricular injection with 14 pM of beta-endorphin, 24 h prior to viral infection, led to an increased fatality of mice previously reconstituted with T-lymphocytes but not in animals receiving only syngeneic NK cells. The increased fatality caused by the neuropeptide was antagonized by naloxone but not beta-endorphin-(1-27). Separation of splenocyte cell populations by buoyant density centrifugation demonstrated that small race lymphocytes, and not the large granular lymphocytes, were responsible for protection of nude mice from the central nervous system infection with ts-VSV. The beta-endorphin-responsive immune cells were shown to be a minor fraction of the small race T-lymphocyte population that bear the asialo-GM1 marker.     

The cellular immunotherapy of primary brain tumors.

The use of adjuvant immunotherapy for the treatment of primary malignant brain tumors dates to studies performed in the 1960's and 1970's using non-specific immune stimulators. Although the theoretical designs have remained similar, recent advances in molecular biotechnology have produced a new group of recombinant cytokines, spawning a new generation of immunotherapy-based clinical trials. In contrast to other published Phase I/II studies, we have had highly encouraging preliminary results using lymphokine-activated killer (LAK) cells and recombinant human Interleukin-2 (rIL-2; Cetus, Emeryville, CA), when the patients' use of corticosteroids could be restricted while on study. Patients with recurrent grade 3/3 glioma received multiple cycles of autologous LAK cells and rIL-2, post-operatively, via an Ommaya reservoir implanted into the tumor cavity following re-operation. The overall median survival for 13 patients with grade 3/3 glioma has not yet been reached at 55 weeks following second surgery, [mean +/- SEM, 64.7 +/- 10.5 weeks], with 5 patients still alive. Three patients have had partial responses (PR) demonstrated by CT scanning. In addition, one patient with grade 2/3 glioma has had a complete response (CR), with the disappearance of all residual CT-documented enhancement and mass effect.     

Analysis of cytolytic activity and cell surface phenotypes of lymphokine activated killer cells stimulated with R-IL 2 and an anti-CD 3 antibody

We analyzed cytolytic activity and cell surface phenotypes of LAK (lymphokine activated killer) cells which were stimulated with recombinant IL-2 and an anti-CD3 antibody. This study involved 9 patients with astrocytomas, one patient with ganglioglioma, and one patient with medulloblastoma and their ages ranged between 5 and 80yr. Peripheral blood lymphocytes were separated from about 40 ml venous blood on a Ficoll-Paque gradient. After PBL's were cultured with r-IL 2 and anti-CD 3 antibody for about two weeks, more than 10(8) LAK cells could be obtained. Their cytolytic activity was measured by standard 4 hours-51Cr release assay and K 562, Daudi, U 251 MG, MCF-7, and autologous tumor cells were used as target cells. Their surface phenotypes (CD 3, CD 4, CD 8, CD 16, CD 25, Leu 7, HLA-DR) were measured by FACS. These LAK cells showed weaker killing activity against all kinds of tumor cells than usual LAK cells (LAK cells stimulated with r-IL 2 alone) did. Their surface phenotypes were more sensitive to CD 3 and less sensitive to CD 16 and Leu 7 than those of usual LAK cells. One of weak points of so-called LAK therapy is that many PBL's have to be obtained by leukapheresis and because of that, it is difficult to undertake LAK therapy to children or elderly patients. However, by using an anti-CD 3 antibody, enough LAK cells as effector of LAK therapy could be obtained more easily.(ABSTRACT TRUNCATED AT 250 WORDS)     

组胺诱导胶质瘤原代培养细胞凋亡的实验研究

目的观察组胺诱导人脑胶质瘤原代培养细胞凋亡的作用，探讨组胺诱导人脑胶质瘤原代培养细胞凋亡与钙离子内流之间的关系。方法取手术中获得的新鲜脑胶质瘤组织，加入含10％小牛血清的1640培养液中培养；取生长良好的细胞分别加入含0．01mg／ml，0．05mg／ml和0．1mg／ml不同浓度组胺的培养液，进行细胞的原代培养。激光共聚焦显微镜检测细胞膜内游离钙离子浓度；流式细胞仪检测不同浓度组胺作用下细胞的凋亡率。结果加入含0.05mg／ml和0.1mg／ml组胺培养液的人脑胶质瘤原代培养细胞内游离钙离子浓度明显升高，诱导细胞凋亡，细胞凋亡的发生在一定范围内与加入的组胺呈剂量、时间依赖关系。结论组胺通过增加人脑胶质瘤原代培养细胞内游离钙离子浓度诱导细胞凋亡的发生。     

Lymphokine-activated killer (LAK) and adherent LAK (A-LAK) activity in multiple sclerosis

The cytotoxic activity of lymphokine-activated killer (LAK) cells against enriched cultures of oligodendrocytes (OL) was investigated in multiple sclerosis (MS) and controls. Human LAK cells, derived from macrophage-depleted peripheral blood mononuclear cells (PBMC) and incubated with recombinant human interleukin-2 (IL-2) (20-80 U/ml), mediated high levels of cytotoxicity against Raji cells but low levels of cytotoxicity against primary cultures of bovine OL. Cytotoxicity was not enhanced by incubation with a high level of IL-2 (500 U/ml). No statistically significant differences in LAK cell activity against bovine OL were observed among the study groups. Enriched adherent LAK (A-LAK) cells mediated greater levels of cytotoxicity against both bovine OL and tumor cell lines than unfractionated LAK cells. Flow cytometric analysis indicated that A-LAK effector cells were CD4-, CD8+, and CD16+. Furthermore, A-LAK cells mediated lysis of OL derived from several different animal species. Our results suggest that LAK and A-LAK cells can mediate cytolysis of OL in culture similar to that observed with a number of different tumor cell lines. However, no significant difference in cytolysis was identified between MS and control groups in this study.     

An immune cell population that responds to β-endorphin and is responsible for protecting nude mice from the fatal consequences of a virus infection of the central nervous system

Reconstitution of 3- to 4-week-old BALB/c nude (nu/nu) mice with 10⁷ syngeneic splenocytes, 48 h before intracerebral inoculation with a temperature-sensitive (ts) mutant of VSV (tsG31 KS5), provided protection from the fatal consequences of clinical disease in 80–90% of the infected animals. Reconstitution of animals with 10⁷ splenocytes, first depleted of natural killer (NK) cells with anti-asialo G_M1 and complement, also afforded protection against the infectious disease. Depletion of T-lymphocytes with anti-thy-1.2 antibody and complement, however, provided little protection with approximately 40% of the animals succumbing to the virus infection within 30 days post-infection. A single intracerebroventricular injection with 14 pM of β-endorphin, 24 h prior to viral infection, led to an increased fatality of mice previously reconstituted with T-lymphocytes but not in animals receiving only syngeneic NK cells. The increased fatality caused by the neuropeptide was antagonized by naloxone but not β-endorphin-(1–27). Separation of splenocyte cell populations by bouyant density centrifugation demonstrated that small race lymphocytes, and not the large granular lymphocytes, were responsible for protection of nude mice from the central nervous system infection with ts-VSV. The β-endorphin-responsive immune cells were shown to be a minor fraction of the small race T-lymphocyte population that bear the asialo-G_M1 marker.     

Activated natural killer cells adhere to cultured hippocampal neurons and affect the dendritic morphology

To examine the manner of interactions between immune cells and central nervous system (CNS) neurons, mouse hippocampal neurons were co-cultured with lymphokine (IL-2)-activated killer (LAK) cells. Immunocytochemical and time-lapse observations indicated that LAK cells migrated along neuronal processes and made adhesive contacts with them. In addition to the direct physical effects, LAK cells released glutamate, induced the formation of beads-like structure in the dendrites of about 14% of hippocampal neurons and caused the reduction of dendritic protrusions. These results suggest that infiltrating immune cells can form direct adhesive connections with CNS neurons and affect their dendritic morphology.     

Targeting therapy for glioma by LAK cells coupled with bispecific antibodies

Targeting of T cells or natural killer (NK) cells to tumours by using bispecific antibodies has attracted increasing interest in the past few years. We treated 31 patients with malignant glioma using adoptive transfer of lymphokine-activated killer (LAK) cells coupled to a bispecific antibody (anti-CD3+anti-glioma) as post-operative adjuvant therapy. Although this study excluded patients with deeply-seated tumours or a poor performance status, approximately 50% of the patients remained alive after 3 years, and 40% were free of recurrence. Serial CT scans revealed disappearance of remnant tumours in some patients. In addition, CT-guided stereotactic biopsy of the tumour in 3 patients showed extensive necrosis and degeneration after specific targeting therapy (STT). Five patients developed acute infection, and one of them died of bacterial meningitis. Our results suggest that antibody-targeted LAK therapy can achieve a higher response rate in patients with standard LAK therapy or any type of conventional treatment.