Inhibition by Fibrin Coagulation of Lung Cancer Cell Destruction by Human Interleukin-2-activated Killer Cells

We examined the effect of fibrin coagulation on tumor cytotoxicity mediated by human lymphokine (IL-2)-activated killer (LAK) cells. LAK cells were induced from peripheral blood mononuclear cells (MNC) by culture with recombinant IL-2 for 4 or 5 days, and LAK cell-mediated cytotoxicity against tumor cells was assessed by ⁵¹Cr release assay in the presence or absence of plasma from normal subjects and lung cancer patients. Plasma did not affect the phase of induction of LAK activity by IL-2, but dose-dependently inhibited the effector phase of LAK cell-mediated cytotoxicity against Daudi cells. Similar inhibition of LAK cell-mediated cytotoxicity was observed on pretreatment of Daudi cells and human lung cancer cell lines with human fibrinogen plus thrombin. A parallel relationship was found between the amount of fibrinogen in plasma of lung cancer patients and inhibition of LAK cytotoxicity. This inhibition was reduced by addition of anticoagulants (heparin or argatroban). These findings suggest that fibrin coagulation on tumor cells protects them from LAK cell-mediated tumor cytotoxicity in malignant lesions and that a combination of an anticoagulant drug and IL-2/LAK therapy may be effective for treatment of lung cancer patients.     

Interleukin-18 Synergism with Interleukin-2 in Cytotoxicity and NKG2D Expression of Human Natural Killer Cells

Natural killer (NK) cells play an important role in anti-tumor immunity. Interleukin (IL)-18 is animmunoregulatory cytokine that induces potent NK cell-dependent anti-tumor responses when administratedwith other cytokines. In this study, we explored the effects of combining IL-18 and IL-2 on NK cytotoxicity aswell as expression levels of the NK cell receptor NKG2D in vitro. Freshly isolated PBMCs were incubated for48 h with IL-18 and IL-2, then CD107a expression on CD3-CD56+ NK cells was determined by three-colour flowcytometry to evaluate the cytotoxicity of NK cells against human erythroleukemia K562 cells and human coloncarcinoma HT29 cells. Flow cytometric analysis was also employed to determine NKG2D expression on NK cells.The combined use of IL-18 and IL-2 significantly increased CD107a expression on NK cells compared with usingIL-18 or IL-2 alone, suggesting that the combination of these two cytokines exerted synergistic enhancement ofNK cytotoxicity. IL-18 also enhanced NKG2D expression on NK cells when administered with IL-2. In addition,blockade of NKG2D signaling with NKG2D-blocking antibody attenuated the up-regulatory effect of combiningIL-18 and IL-2 on NK cytolysis. Our data revealed that IL-18 synergized with IL-2 to dramatically enhance thecytolytic activity of human NK cells in a NKG2D-dependent manner. The results appear encouraging for theuse of combined IL-18 and IL-2 in tumor immunotherapy.     

Induction by Interleukin-15 of Human Killer Cell Activity against Lung Cancer Cell Lines and Its Regulatory Mechanisms

Interleukin (IL)-15 is a novel cytokine with IL-2-like activity. In the present study, we examined IL-15-mediated induction of killer activity of peripheral blood mononuclear cells (MNC) against lung cancer cell lines, and the regulatory mechanisms of this induction by IL-15. Cytotoxic activity was measured by ^51Cr release assay. IL-15 at concentrations of more than 10 ng/ml induced significant killer activity of blood MNC against a small cell lung cancer cell line (SBC-3), as well as Daudi cells, and 50 ng/ml was considered its optimal concentration. A time course study revealed that an incubation period of 4–6 days was optimal for induction of killer activity. MNC cultured with IL-15 also exhibited killer activity against other lung cancer cell lines (H-69, N-291 and PC-9 cells). IL-15 and IL-12 had additive effects on induction of killer activity against SBC-3 cells. On the other hand, IL-15 had no synergistic or additive effect on induction of killer activity by IL-2. Fresh human monocytes isolated by centrifugal elutriation augmented the development of killer activity of lymphocytes stimulated by IL-15. As a humoral regulatory factor, IL-4 had a suppressive effect on induction of killer activity by IL-15. IFN-γ, IL-1β, TNF-α, IL-6 or IL-10 had no effect on induction of killer activity by IL-15 at the optimal concentration. These results suggest that IL-15 has potential for the immunotherapy of ling cancer.     

应用人胎儿脾LAK细胞治疗62例晚期恶性肿瘤病人的疗效观察

采用人胎儿脾ＬＡＫ细胞治疗６２例晚期恶性肿瘤病人。结果表明人胎儿脾ＬＡＫ细胞的临床应用安全可行，多数病人在治疗期间一般状况得到改善。总缓解率为（ＰＲ）２２．５８％，平均缓解时间为１５．９５个月。缓解率与克氏评分和接受４次疗程的ＩＬ－２／ＬＡＫ细胞用量有关。在１８．４个月的治疗期间，死亡率为２０．９７％，平均生存期为１６．５０个月。ＩＬ－２／ＬＡＫ细胞对肾癌、黑色素瘤、结肠癌、肺癌和肝癌的转移灶有较好疗效，对原发灶也有一定作用。     

Co-production of Interleukin-1 and Interleukin-6 in Tumor Cell Lines Elaborating Colony-stimulating Factors

Six carcinoma cell lines elaborating colony-stimulating factors (CSFs) were examined by enzyme-linked immtmosorbent assay and Northern blotting to determine whether or not they co-produced interleukin-lα (IL-lα), IL-lβ and IL-6. All 6 cell lines were co-producers; IL-lα was produced in all 6, IL-6 in 5 and IL-1β in 3 of them. These results indicate that IL-1 and IL-6 are commonly co-produced in CSF-producing tumors.     

Cell Cycle Assessment of Adoptive Transferred Lymphokine Activated Killer Cells

The use of lymphokine activated killer (LAK) cells for adoptivetransfer therapy has been reported from number of clinical trials.To our knowledge, however, there has been no report concerningthe cell cycle progression of LAK cells. Thus, for the presentstudy we have attempted to examine the LAK cell cycle eitherbefore or after transfer. In vitro and in vivo analyses of LAKcells labeled with bromodeoxyuridine (BrdU) were carried outusing two-parameter flow cytometries of their nuclear stainingusing fluorescein isothiocyanate(FITC)- conjugated anti-BrdUantibody and propidium iodide (PI). The in vitro growth of BrdU-positivecells showed the cells to divide once in the S phase, continueto the G₂-M phase and return to the G₀-G₁ phase with a similarpattern after 48 or 72 h culture. They formed a definite subpopulationand were of phenotypes, thy-1.2 (+), Lyt-1.1 ( – ), Lyt-2.1(+), L3T4 ( – ) and AGM1 (+). The percentage of BrdU-positivecells decreased significantly (P< 0.05) when treated withcomplement plus anti-thy-1.2, anti-Lyt-2.1 or anti-AGM1 anti-bodies, demonstrating BrdU-labeled LAK cells to have the samephenotype as control LAK cells. As in vitro, the in vito cellcycle of LAK cells 48 and 72 h after transfer had a similarpattern, and the LAK cells also continued on to the S phaseafter the first division. The in vivo growth of the LAK cellstreated with interleukin-2 (IL-2) was promoted 24 and 48 h aftertransfer. In conclusion, the present study showed LAK cellsto be capable of dividing following transfer and to keep theirown phenotypic characteristics; also, that treatment with IL-2may promote their division. Adoptive transfer therapy seemsto be a viable therapeutic method.     

Selective Expansion of Human Natural Killer Cells from Peripheral Blood Mononuclear Cells by the Cell Line, HFWT

An anchorage-dependent Wilnis tumor cell line HFWT was found to stimulate selective and remarkable expansion of human natural killer (NK) cells from human peripheral blood mononuclear cells (PBMC). After PBMC of healthy donors were cultured on irradiated HFWT cells for 10–21 days, the lymphocytes expanded 58- to 401-fold. This NK cell expansion required direct contact of PBMC with live, but not fixed, HFWT cells. The PBMC from an end-stage brain tumor patient also expanded 156-fold, whereas those cultured with irradiated NK-sensitive K562 grew only 30.5-fold. CD16⁺CD56⁺ NK cells accounted for more than 70% of the population expanded on HFWT cells. No essential difference in expression of NK receptors was observed in the expanded NK cells on HFWT and K562 and without feeder cells. The expanded NK cells killed not only fresh HFWT cells but, unexpectedly, also MHC class I-expressing autologous brain tumor cells at an effector/target ratio of 4 for 24 h. These results will contribute to the development of a large-scale preparation method for human NK cells, which will aid studies of NK cell biology and possible treatment of brain tumors.     

CIK细胞对一线化疗后晚期非小细胞肺癌维持治疗的疗效分析

目的 探讨细胞因子诱导的杀伤细胞(CIK)维持治疗一线化疗后晚期非小细胞肺癌(NSCLC)的疗效及安全性.方法 收集96例ⅢB~Ⅳ非小细胞肺癌患者,一线治疗为4~6个周期的含铂两药方案化疗,将疗效达稳定或以上的患者随机分两组,一组给予CIK免疫治疗,一组给予支持治疗,比较两组的PFS、有效率、外周血T淋巴细胞亚群、KPS评分及评估安全性.结果 CIK组和对照组的中位PFS分别为8.0个月(95%CI 7.3~8.7)和6.0个月(95%CI 4.8~7.2)(P=0.002).CIK组疾病控制率为81.25%,显著高于对照组的62.50%(P<0.05).CIK治疗组患者外周血CD3+、CD3+/CD4+细胞比率较治疗前显著上升,对照组T淋巴细胞亚群没有变化.CIK细胞治疗后患者KPS评分为(71.53±7.96)分,较治疗前显著提高(P<0.05).CIK治疗组中3例出现一过性发热,3例皮疹,2例肌肉痛.结论 CIK维持治疗一线化疗后稳定的晚期非小细胞肺癌患者,可提高PFS及近期疾病控制率,改善免疫功能及生活质量,安全性好.     

Two Populations of Mouse Lymphokine-activated Killer Cells Separated by Use of Soybean Agglutinin

We separated lymphokine-activated killer (LAK) cells induced from spleen cells of BALB/c mice by culturing with recombinant interleukin-2 (rIL-2) into soybean agglutinin-positive (SBA⁺) and soybean agglutinin-negative (SBA^-) fractions with a cell sorter at the time when LAK activity reached a maximum (day 3). We found that the cells with LAK activity were enriched in the SBA⁺ fraction. Analysis of cell surface phenotypes revealed that the SBA⁺ cells are of non-T cell origin, while the SBA^- fraction consists of T cells. We also found that the large granular lymphocyte (LGL) fraction of spleen cells obtained with a Percoll gradient became SBA⁺ after culture for 3 days with rIL-2, whereas cells of high density did not. The change in SBA binding sites was also examined on C57BL/6 mouse spleen cells and we found that NK1.1⁺ non-T cells selectively acquire SBA binding sites at an early stage of activation with rIL-2. On the other hand, sorted SBA^- cells gradually acquired SBA binding sites on extended culturing with rIL-2. These results suggest that the expression of SBA binding sites is related to the stage of cell activation with rIL-2, though cells of the NK-lineage became SBA⁺ at an earlier period of a culture with rIL-2 than cells of the T-lineage. Utilizing these findings, we separated NK-derived LAK cells by means of SBA-binding, and used the separated cells for adoptive immunotherapy for experimental pulmonary metastasis. We found that SBA⁺-NK cell-derived LAK cells showed stronger activity for the inhibition of experimental pulmonary metastasis than SBA -T cell-derived LAK cells.     

抗原致敏树突状细胞诱导CIK对肺癌细胞杀伤作用的研究

[目的]研究肿瘤抗原致敏的树突状细胞（DC）诱导淋巴因子激活的杀伤细胞（LAK）和细胞因子诱导的杀伤细胞（CIK）对肺癌细胞株A549和肺腺癌原代细胞的杀伤作用。[方法]取健康人外周血单个核细胞,常规诱导出DC、CIK、LAK细胞。用肺癌A549细胞提取的肿瘤抗原冲击DC,倒置显微镜下观察DC形态。流式细胞仪检测DC经抗原冲击和未经抗原冲击后其表型变化。LDH释放法测定杀伤活性。[结果]DC经肿瘤抗原冲击后在镜下呈典型成熟形态,其表面分子CD40、CD80、CD86和HLA-DR的表达明显较未经肿瘤抗原冲击的DC高。DC＋CIK细胞对A549和肺腺癌原代细胞的杀伤活性高于CIK细胞、LAK细胞和DC＋LAK细胞（P〈0.05）,随着效靶比的升高,其杀伤效应随之增强（P〈0.05）。[结论]肿瘤抗原致敏的DC可诱导特异性CIK细胞,DC＋CIK细胞对A549和肺腺癌原代细胞的杀伤作用明显高于DC＋LAK、CIK、LAK细胞。     

CIK细胞对晚期非小细胞肺癌预后的影响

目的观察细胞因子诱导的杀伤细胞（CIK细胞）体外培养后,对晚期非小细胞肺癌的疗效。方法收集经确诊的、并发生转移、且采用标准治疗方案的非小细胞肺癌患者20例作为观察组,取外周血分离单个核细胞（PBMC）,体外细胞因子诱导培养CIK细胞,流式细胞仪检测细胞表型。20例患者均接受CIK细胞免疫治疗,观察其免疫活性及生存期。并以20例采用标准治疗方案而未经CIK治疗的晚期非小细胞肺癌患者作为对照组。结果观察组培养后的细胞表型CD3、CD3＋CD56＋明显升高,生存期为（16.15±3.8）个月,对照组生存期为（9.12±3.2）个月。结论经CIK细胞治疗后晚期非小细胞肺癌患者的生存期明显延长。     

Generation and Characterization of Lymphokine-activated Killer Cells against Fresh Human Leukemia Cells

Lymphokine-activated killer (LAK) cells generated from 15 acute leukemia patients in remission showed significant levels of cytotoxicity against Daudi 1A4, a natural killer-resistant cell line. This indicates that lymphocytes of leukemia patients in remission could respond to interleukin-2 to generate conventional LAK cells. However, LAK cells caused lysis of autologous leukemia cells at considerably lower levels in seven out of the 15 patients, with the exception of one case (48.6% cytolysis). None of the remaining eight patients exhibited LAK activity against autologous leukemia cells. On the other hand, patients' LAK could lyse allogeneic leukemia cells including those resistant to autologous LAK. Thus, patients' LAK seem not to be defective in lysis of leukemia cells. In the cold target competition analysis, the binding of patients' LAK to leukemia cells could be inhibited by autologous and allogeneic leukemia cell competitors, implying that almost all leukemia cells could be recognized by patients' LAK. Most LAK cells from normal donors showed significant lysis of allogeneic leukemia cells, but some leukemia cells were found to be resistant to lysis. LAK cells against both leukemia cells and Daudi 1A4 were phenotypically heterogenous, and were predominantly observed in the T3⁻ fraction in the precursor phase. In the effector phase, whereas LAK activity against leukemia cells was also predominantly shown in the T cell-depleted fraction, similar levels of LAK activity against Daudi 1A4 were found in both the T cell-depleted and -enriched fractions.     

An Interleukin-15 Superagonist Enables Antitumor Efficacy of Natural Killer Cells Against All Molecular Variants of SCLC

IntroductionSCLC is a highly aggressive tumor with a 5-year survival rate of less than 6%. A heterogeneous disease, SCLC is classified into four subtypes that include tumors with neuroendocrine and non-neuroendocrine features. Immune checkpoint blockade has been recently added for the frontline treatment of SCLC; however, this therapy has only led to modest clinical improvements. The lack of clinical benefit in a cancer type known to have a high tumor mutational burden has been attributed to poor T-cell infiltration and low expression of MHC-class I in most SCLC tumors. In an attempt to devise a more effective immunotherapeutic regimen, this study investigated an alternate approach on the basis of the use of the clinical-stage interleukin-15 superagonist, N-803.MethodsPreclinical models of SCLC spanning all molecular subtypes were used to evaluate the susceptibility of SCLC to natural killer (NK)-mediated lysis in vitro, including NK cells activated by N-803. Antitumor activity of N-803 was evaluated in vivo with a xenograft model of SCLC.ResultsIn vitro and in vivo data revealed differences in susceptibility of SCLC subtypes to lysis by NK cells and that NK cells activated by N-803 effectively lyse SCLC tumor cells across all variant subtypes, regardless of their expression of MHC-class I.ConclusionsThese findings highlight the potential of a novel immune-based intervention using a cytokine-based therapeutic option for the treatment of SCLC. We hypothesize that N-803 may provide benefit to most patients with SCLC, including those with immunologically cold tumors lacking MHC expression.     

晚期非小细胞肺癌患者外周血DC亚型与 NK细胞的关系

 目的 探讨非小细胞肺癌患者外周血树突状细胞亚群（DC1/DC2）和机体NK细胞含量之间的关系及其临床意义。方法 采用流式细胞术检测40例肺癌患者外周血DC亚群（CD11c+DC/DC1和 CD123+ DC／DC2）、NK细胞含量及血浆IL 12的浓度， 并以10例健康受试者作为对照。结果 肺癌患者外周血CD11c+DC百分率为（0.66±0.24）％，比对照组（1.38±0.18）％明显降低（P<0.01），CD123+ DC百分率（0.28±0.17）％与对照组（0.27±0.11）％相比， 差异无统计学意义（P＞0.05）；肺癌患者与健康人比较，NK细胞含量及IL 12的浓度均降低（P<0.05）。NSCLC 患者NK细胞的含量与CD11c+百分数及血浆IL 12 浓度均呈正相关（P<0.05），与CD123+百分数呈负相关（P<0.01）。DC各亚型百分率同患者的卡氏评分、化疗史有关联（P<0.01），NK细胞的含量与年龄相关（P<0.05）。结论 非小细胞肺癌患者DC1功能低下，IL 12分泌能力障碍，从而影响了NK细胞的活性。DC亚型与NK细胞含量之间以及它们与临床生物学行为之间都有一定关系。     

Dipyridamole Combined with Tumor Necrosis Factor-α Enhances Inhibition of Proliferation in Human Tumor Cell Lines

In the search for cytokines whose antiproliferative action could be enhanced by combination with dipyridamole, 2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido[5,4,d]pyrimidine, the combination of tumor necrosis factor-α (TNF-α) with this agent was evaluated in various human tumor cell lines. Inhibition of the proliferation of human melanoma cell lines MM-1CB and HMV-1 by TNF-α (1–10² U/ml) was enhanced in culture dishes by combination treatment with dipyridamole (0.1–10 μM). The enhancement effect was also detected in other tumor cell lines: T9S (glioma), SCC-1CB (squamous cell carcinoma), HAC-2 (ovarian clear-cell carcinoma), HLE (hepatoma), HEC-1 (endometrial adenocarcinoma) and HOC-21 (ovarian serous cystadenocarcinoma). The incorporation of [¹⁴C]amino acids and [³H]nridine into acid-insoluble cell materials in the combination-treated cells was not significantly different from that in cells treated with TNF-α or dipyridamole. However, the incorporation of [³H]thymidine was specifically inhibited in all cell lines examined after more than 12 h of the TNF-α and dipyridamole combination treatment, although neither agent alone inhibited this incorporation. On the other band, the growth of tumors induced by the injection of MM-1CB and HMV-1 cells into nude mice was more markedly inhibited by the subcutaneous administration of TNF-α in combination with orally administered dipyridamole than by either agent alone. The results presented suggested that dipyridamole is beneficial in assuring the effectiveness of anti-cancer cytokine therapy.     

Antitumor Activity of Lentivirus-mediated Interleukin -12 Gene Modified Dendritic Cells in Human Lung Cancer in Vitro

Objectives: Dendritic cell (DC)-based tumor immunotherapy needs an immunogenic tumor associatedantigen (TAA) and an effective approach for its presentation to lymphocytes. In this study we explored whethertransduction of DCs with lentiviruses (LVs) expressing the human interleukin-12 gene could stimulate antigenspecificcytotoxic T cells (CTLs) against human lung cancer cells in vitro. Methods: Peripheral blood monocytederivedDCs were transduced with a lentiviral vector encoding human IL-12 gene (LV-12). The anticipated targetof the human IL-12 gene was detected by RT-PCR. The concentration of IL-12 in the culture supernatant of DCswas measured by ELISA.Transduction efficiencies and CD83 phenotypes of DCs were assessed by flow cytometry.DCs were pulsed with tumor antigen of lung cancer cells (DC+Ag) and transduced with LV-12 (DC-LV-12+Ag).Stimulation of T lymphocyte proliferation by DCs and activation of cytotoxic T-lymphocytes (CTL) stimulatedby LV-12 transduced DCs pulsed with tumor antigen against A549 lung cancer cells were assessed with methylthiazolyltetrazolium (MTT). Results: A recombinant lentivirus expressing the IL-12 gene was successfullyconstructed. DC transduced with LV-12 produced higher levels of IL-12 and expressed higher levels of CD83than non-transduced. The DC modified by interleukin -12 gene and pulsed with tumor antigen demonstratedgood stimulation of lymphocyte proliferation, induction of antigen-specific cytotoxic T lymphocytes and antitumoreffects. Conclusions: Dendritic cells transduced with a lentivirus-mediated interleukin-12 gene have anenhanced ability to kill lung cancer cells through promoting T lymphocyte proliferation and cytotoxicity.     

Augmentation of Murine Lymphokine-activated Killer Cell Induction by a Factor Produced by Nocardia rubra Cell Wall Skeleton-stimulated T Cells

Four-hour exposure of C3H/HeN mouse spleen cells to Nocardia rubra cell wall skeleton (N-CWS) before 4-day culture with a suboptimal dose of human recombinant interleukin 2 (rIL 2) augmented the induction of lymphokine-activated killer (LAK) cell activity, whereas the treatment with N-CWS alone induced no cytotoxicity. In accordance with this, the IL 2 binding activity of spleen cells was augmented by combined stimulation with N-CWS and rIL 2. The augmented cytotoxicity was mediated by Thy-1.2⁺, Lyt-1.1⁻, Lyt-2.1⁻ and asialo GM₁⁺ cells. Cell cultures in diffusion chambers revealed that N-CWS-treated spleen cells produced a LAK cell induction-helper factor (LAK-helper factor, LHF) when cultured with rIL 2. The LHF production required Thy-1.2⁺, Lyt-1.1⁺, Lyt-2.1⁺ and asialo GM₁⁻ cells, and the coexistence of unstimulated accessory cells was also essential for the LHF production. Cells responding to both LHF and rIL 2 to generate LAK activity were Thy-1.2⁻, Lyt-1.1⁻, Lyt-2.1⁻ and asialo GM₁⁺. The culture fluid of spleen cells stimulated with both N-CWS and rIL 2 contained no tumor necrosis factor (TNF) activity, and the additional stimulation with N-CWS caused no production of either IL 2 or interferon (IFN). Murine recombinant interleukin la (Mu-rIL 1α) could not replace the augmentative effect of N-CWS on LAK cell induction. These results suggest that in the presence of rIL 2, N-CWS stimulates murine T cells to produce LHF that is probably distinct from IL 1, IL 2, TNF and IFN.     

Antitumor Effect of Recombinant Human Interleukin-2 on the Growth of Murine Hemangioendothelioma D14 in Nude Mice: Occurrence of Large Granular Cells in the Tumor

The antitumor effect of recombinant human interleukin-2 (rIL-2) on murine hemangioendothelioma D14 (D14) in female BALB/c-nu/nu mice was examined histologically. D14 cells which had been maintained in vitro were transplanted subcutaneously into nude mice on day 0 (1 × 10⁷ cells/mouse). The mice with established tumor on day 28 received rIL-2 subcutaneously at a dose of 20 μg/mouse/ day for 35 days. On day 63, the mice were killed, and the tumor, spleen and bone marrow were examined histologically. In the mice that had received rIL-2, tumor growth was significantly suppressed. Histologically, there was marked infiltration of large granular cells (about 15-30 μ in diameter) in the tumors. In the adjacent areas, there was a significant increase in the number of tumor cells showing karyorrhexis. The large granular cells (LGC) contained periodic acid Schiff-positive round granules in the cytoplasm and were stained positively for Thy-1.2 surface antigen. The LGC were also positive for asialo GM1 surface antigen but not for Lyt-1, Lyt-2 or IgG surface antigens. This evidence suggests that the LGC are lymphokine-activated killer-like cells which were derived from a natural killer cell lineage. The concomitant increases in the number of LGC and the number of cells showing karyorrhexis in the tumors of the mice treated with rIL-2 suggest that LGC play an important role in the destruction of tumor cells.     

NK细胞对人鼻咽癌细胞CNE2裸鼠皮下移植瘤的抑制作用

 目的 研究同种异体NK细胞对人鼻咽癌细胞（CNE2）裸鼠皮下移植瘤的抑制作用。方法PCR-SSP法检测CNE2细胞HLA-A、B、Cw表型、NK细胞KIR表型（选择3例健康者为试验对象）,磁珠分离法分离NK细胞并进行体外培养扩增,LDH释放法测定NK细胞对CNE2细胞的体外杀伤活性。12只BALB/c裸鼠分为两组,每组6只,对照组裸鼠每只皮下接种1×106CNE2细胞,治疗组裸鼠每只皮下接种1×106CNE2细胞,同时每只经尾静脉注入3×107NK细胞,观察两组裸鼠成瘤时间、成瘤率、肿瘤体积变化、计算抑瘤率。结果 CNE2细胞表面HLA-A、B、Cw表型为A2,24;B18,35;Cw4,7,3例健康者均表达KIR2DL1、KIR2DL3、KIR3DL1、KIR3DL2。效靶比5∶1、10∶1、20∶1、30∶1时,NK细胞对CNE2细胞的杀伤活性分别为（9.37±2.14）%、（27.14±1.82）%、（36.40±4.28）%and（54.67±2.80）%。对照组和NK细胞治疗组肿瘤出现时间分别为（10.00±2.68）d、（18.80±1.64）d,（P〈0.01）,成瘤率分别为100%（6/6）、83.33%（5/6）,对照组和NK细胞治疗组裸鼠的瘤重分别为（2.22±0.09）g、（1.42±0.09）g,（P〈0.01）,NK治疗组的抑瘤率为36.04%。肿瘤组织石蜡切片病理学鉴定为低分化鳞状上皮细胞癌,NK细胞治疗组可见角化肿瘤细胞,较多的淋巴细胞浸润和大量细胞坏死区。结论 NK细胞对鼻咽癌裸鼠皮下移植瘤有明显的抑制作用,有希望成为治疗鼻咽癌的新方法。     

Inhibition by Sphingosine of Leukemic Cell Killing by Human Monocytes Activated with Interleukin-2: A Possible Role of Protein Kinase C

Sphingosine and its analogs, which inhibit protein kinase C (PKC), are known to be potent inducers of apoptosis in tumor cells. However, we were concerned that sphingosine might also interfere with anti-tumor cells of the immune system. Therefore, we evaluated the effect of sphingosine on activation of human monocytes by interleukin-2 (IL-2) for killing of leukemic cells. Monocytes, purified by elutriation and adherence, were activated with IL-2 or interferon-gamma (IFN-γ) in the presence or absence of sphingosine or another inhibitor for 18 h. Then the monocytes were washed and the culture medium was replaced with fresh medium to remove the sphingosine. HL-60 and K562 leukemic cells were added to the monocyte cultures. Over the next 48 h, the cytotoxic activity of the monocytes towards the leukemic cells was assessed by means of an ¹¹¹indium-releasing assay. IL-2-activated monocytes lysed 48±3% of HL-60 cells and 44±3% of K562 cells. Sphingosine, dihydrosphingosine, N, N-dimethylsphingosine, and the PKC inhibitor H7 inhibited the activation of monocytes by IL-2, blocking cytotoxic activity against the leukemic cells by approximately 75%. These inhibitors were not toxic to monocytes at the concentrations used. In a PKC assay, sphingosine and H7 inhibited PKC activity in IL-2-treated monocytes. Thus, sphingosines, by inhibiting PKC activity, inhibited activation of monocytes by IL-2, which inhibited the killing of leukemic cells.