Recombinant gamma interferon provokes resistance of human breast cancer cells to spontaneous and IL-2 activated non-MHC restricted cytotoxicity

Natural and lymphokine activated killer cells (NK and LAK) are believed to play an important role in the control of tumour progression and metastasis. Their specific receptors on tumours cells are still unknown. Several studies suggest that these cells recognise and eliminate abnormal cells with deleted or reduced expression of MHC class I molecules. Previous reports suggest that interferons (IFN), by increasing MHC class I expression on target cells, induce resistance to killing by NK cells. We investigated the role of MHC molecule expression by two human breast cancer cell lines T47D and ZR75-1 in their susceptibility to NK and LAK cells. These two cell lines spontaneously express low levels of HLA class I antigens but no HLA class II molecules. After IFN-gamma treatment they both overexpressed MHC class I and de novo expressed class II molecules as detected by flow cytometry, quantified by a radioimmunoassay and analysed by two-dimensional gel electrophoresis. Opposed to untreated cells these IFN-gamma treated cells were resistant to NK and LAK lysis. Furthermore, preincubation of IFN-gamma treated breast cancer cells with F(ab')2 fragments of monoclonal antibodies to HLA class I and HLA class II molecules was unable to restore lysis. In contrast, several complete monoclonal antibodies including anti-HLA class I and HLA class II induced the lysis of target cells whether or not they had been treated by IFN-gamma. The therapeutic use of monoclonal antibodies directed against antigens expressed on tumour cells (ADCC) in conjunction with interferon therapy should be discussed in lymphokine-based strategies for treatment of cancer patients.     

Ewing's sarcoma: ex vivo sensitivity towards natural and lymphokine-activated killing

We studied the ex vivo cell-mediated cytotoxicity of natural killer (NK) and lymphokine-activated killer (LAK) cells against continuously cultured Ewing's sarcoma cells from 3 different patients. Target cell lysis was measured in a 4-hour 51Cr radioisotope release assay. At an effector to target (E:T) ratio of 50:1, the mean (+/- 1 SD) cytolysis by fresh purified large granular lymphocytes (NK cells) was 20 +/- 8, 25 +/- 2, and 21 +/- 3% in Ewing's sarcoma cell lines 6647, 5838, and A4573, respectively. Under identical conditions, NK cells lysed 56 +/- 7% of K562 (a standard NK target), and 3 +/- 3% of Daudi (a standard NK-resistant LAK target). When compared to fresh unseparated peripheral blood mononuclear cells (PBMC), purified NK cells did not exhibit an enhanced cytotoxic reactivity against either Ewing's sarcoma target. In contrast, LAK cells (i.e., PBMC that were preincubated for 4 days in the presence of rIL-2) were highly cytotoxic against all three Ewing's sarcoma targets. LAK activity was dependent on the concentration of rIL-2 used in PBMC cultures. Optimum cell-mediated toxicity against the standard LAK target Daudi (99 +/- 10% cytolysis at 50:1 E:T ratio) was achieved at rIL-2 concentrations of 1,000 u/ml. LAK cells grown under these conditions were also effective against Ewing's sarcoma cells. At an E:T ratio of 50:1, 86 +/- 16, 85 +/- 16, and 67 +/- 13% inhibition was observed in 6647, 5838, and A4573 cells, respectively, as compared to 17 +/- 10, 19 +/- 15, and 29 +/- 11% cytolysis by fresh uninduced PBMC. In summary, our results suggest that rIL-2-induced LAK-type immune effector cells may be of some therapeutic value in the treatment of poor prognosis Ewing's sarcoma.     

Killer cell Ig-like receptors ligand-mismatched, alloreactive natural killer cells lyse primary solid tumors

BACKGROUND: Donor alloreactive natural killer (NK) cells have a potent antileukemic effect in haploidentical stem cell transplantation. Whether alloreactive NK cells are able to specifically kill fresh tumor cells from primary solid tumors was analyzed. METHODS: NK cells were purified from healthy donors for the expression of inhibitory killer cell immunoglobulin (Ig)-like receptors (KIRs), ex vivo expanded, and used as effector cells. Their cytotoxic effect on tumor cells freshly obtained from surgical specimens was assessed by means of a single-cell cytotoxic assay (SCCA). RESULTS: Tumor cells from 1 ovarian, 1 gastric, 3 colon, and 4 renal cell cancers were analyzed and found susceptible to alloreactive NK cell killing (>20% lysis at an effector cell to target cell [E:T] ratio of 10:1 for tumor cells not expressing at least 1 human lymphocyte antigen [HLA] class I KIR-ligand group). Remarkably, NK cells that recognized specific HLA-C group mismatches were able to kill HLA-C KIR ligand-mismatched tumor cells, whereas no lysis of target cells occurred with KIR ligand-matched tumor targets. CONCLUSIONS: Alloreactive NK-cell mediated antitumor effects might provide useful insights for designing new cell therapy approaches against solid tumors.     

Human ovarian carcinoma: evidence for patient-related differences in susceptibility to cytotoxic effectors that attack different cellular subpopulations within a tumour

Human ovarian carcinoma cells obtained from ascites were tested for susceptibility to lysis by peripheral blood NK cells, alpha-interferon-activated NK cells, and interleukin 2-activated killer cells. Cryopreserved tumour cell preparations were used to allow repeated testing of the same target, and the tumour cells were fractionated using albumin density gradients to determine if fractions containing clonogenic (stem) cells were killed. Four tumour cell donors were studied and each showed a different pattern of susceptibility of unfractionated tumour to lysis by different effector cells. Using fractionated tumour cells, we found that NK and interferon-activated NK cells did not always lyse cells in the clonogenic fractions and that interferon activation could in some cases shift killing away from the clonogenic fractions and towards the peak of proliferating (but not self-renewing) colony forming cells. Interleukin 2-activated killer cells (LAK) however, killed the fractions containing clonogenic cells in all 4 cases. The magnitude of killing seen when fractions of the original tumour were tested were often striking when compared to lysis of the unfractionated cells. Apparent heterogeneity between patients and stem cell susceptibility to effector cells may be important determinants of the efficacy of treatment of patients with biologic response modifiers such as interferon and interleukin 2.     

LAK1 antigen defines two distinct subsets among human tumour infiltrating lymphocytes.

Both lymphokine activated killer (LAK) cells and specific cytotoxic T lymphocytes appear to play a role in tumour immunity. Tumour infiltrating lymphocytes (TIL) which display a CD56+ phenotype (both CD3+ and CD3-) are also likely to possess anti-tumour activity. We have previously described a 120 kDa surface antigen, termed LAK1, expressed on a subset of human peripheral blood lymphocytes (20-50%) with both NK and LAK activity. The aim of the present study was to determine whether LAK1 antigen is able to distinguish among TIL two populations of effector cells displaying either specific or non MHC-restricted (NK/LAK) activity. We showed that about 25% of freshly derived TIL were weakly stained with anti-LAK1 monoclonal antibody and most of them were also CD3+ CD56-. After culture in recombinant interleukin-2 the majority of TIL were CD3+ CD56- and the percentage of LAK1+ cells increased up to 50%. Among cloned TIL, only those lacking LAK1 antigen displayed a specific cytotoxicity against the autologous tumour, whereas the non-lytic clones were able to produce both tumour necrosis factor and gamma-interferon. Moreover, when TIL from a renal cell carcinoma were fractionated into LAK1- and LAK1+ populations, the specific lytic activity was mainly evident when LAK1- lymphocytes were used as effector cells. Conversely, LAK activity was confined to the LAK1+ subset.     

Preparation of NK-enriched LAK cells--their potential cytotoxic and ADCC activities

We examined several culture methods to induce proliferation of natural killer (NK) cells from peripheral blood mononuclear cells (PBMC). In the presence of IL-2, a remarkable proliferation of NK cells was observed when PBMC were co-cultured with MMC-treated K562, which is known as a highly sensitive in vitro target cell for the NK assay. Addition of OK-432 or TNF-alpha and IL-1 beta also induced marked NK proliferation in a dose dependent manner. These NK-enriched lymphokine activated killer (LAK) cells showed highly cytotoxic activities against various MHC class I positive or negative tumor cells. They also showed potent ADCC activities against Herceptin-coated SK-BR-3, a HER2/neu positive breast cancer cell line. These results indicated that NK-enriched LAK cells are potent effector cells, and suggested novel therapeutic strategies for nonspecific immunotherapy as well as target immunotherapy in combination with anticancer antibodies, such as Herceptin.     

NKG2D介导NK 细胞对鼻咽癌细胞杀伤作用的体外研究

 目的 探讨鼻咽癌CNE2细胞表面HLA-Ⅰ类分子表型和NKG2D配体的表达情况，进一步了解其对同种异体NK细胞杀伤活性的影响。方法 流式细胞仪检测NKG2D的配体MICA、MICB、ULBP1、ULBP2、ULBP3在K562、CNE2细胞的表达情况。PCR-SSP法分析CNE2细胞HLA-A、B、Cw分型和NK细胞KIR分型。LDH释放法测定5例健康者NK细胞在不同效靶比时对K562、CNE2细胞的杀伤活性，效靶比20：1时观察抗NKG2D配体的单抗对NK细胞杀伤K562、CNE2细胞活性的影响。结果 CNE2细胞表达MICA、MICB、ULBP2，不表达ULBP1、ULBP3。K562细胞表面表达MICA、MICB、ULBP1、ULBP2、ULBP3。5例健康者NK细胞抑制性KIR与CNE2细胞表面的HLA-Ⅰ类分子之间存在错配。效靶比5：1、10：1、20：1、30：1时NK细胞对K562、CNE2细胞的杀伤活性分别为（29．02±0．45）％、（10．50±2．17）％；（44．43±1．36）％、（27．68±1．47）％；（57．82±1．35）％、（36．99±3．13）％：（71．24±2．36）％、（55．00±2．20）％，在各效靶比时NK细胞对K562细胞的杀伤活性较CNE2细胞明显增强（P=0．000）；在效靶比20：1时anti-MICA、anti-MICB、anti-ULBP1、anti—ULBP2、anti-ULBP3可明显抑制NK细胞对K562细胞的杀伤活性，与阻断前相比有显著性差异（P=0．000）；anti—MICA、anti—MICB、anti—ULBP2可明显抑制NK细胞对CNE2细胞的杀伤活性，与阻断前相比有显著性差异（P〈0．01），但anti—ULBP1、anti—ULBP3不能阻断NK细胞对CNE2细胞的杀伤活性。结论 NKG2D配体影响NK细胞对靶细胞的杀伤活性，提高NKG2D配体的表达有可能提高NK细胞的抗肿瘤活性。     

自然杀伤细胞对多发性骨髓瘤细胞株KM-3影响的研究

 目的　研究自然杀伤（NK）细胞对多发性骨髓瘤细胞株KM-3杀伤及诱导凋亡的作用。方法　WST-1法观察不同效靶比的NK细胞对KM-3细胞的杀伤作用;流式细胞术检测Annexin-V+／PI-凋亡细胞、线粒体跨膜电位。结果　NK细胞作用于KM-3细胞后,能显著杀伤KM-3细胞,呈剂量和时间依赖性（P＜0.05）;NK细胞作用于KM-3细胞48 h后,Annexin-V+／PI－细胞比例明显增加,呈剂量依赖性（P＜0.05）;NK细胞作用于KM-3细胞效靶比为10∶1时,Annexin-V+／PI－细胞比例明显增加,呈时间依赖性（P＜0.05）;NK细胞作用于KM-3细胞后,KM-3细胞线粒体跨膜电位明显降低,呈剂量和时间依赖性（P＜0.05）。结论　NK细胞能明显杀伤KM-3细胞并诱导其凋亡,均呈剂量和时间依赖性。     

脐血CD3AK细胞治疗恶性肿瘤的临床应用研究

目的:研究用脐血单个核细胞制备CD3AK细胞的抗肿瘤作用,探讨肿瘤生物治疗近期疗效的免疫指标.方法:分离脐血单个核细胞,分别用IL-2和IL-2 CD3Ab诱导LAK和CD3AK细胞,并测其扩增数量和对K562细胞的杀伤活性;测定肿瘤患者用CD3AK细胞治疗前后外周血T淋巴细胞亚群绝对计数的变化情况和外周血单核细胞(PBMC)的NK杀伤活性.结果:脐血LAK细胞和脐血CD3AK细胞均于培养后11天时扩增倍数最高,分别是培养前的18倍、24倍,对K562的杀伤活性分别是培养前的2.6倍和3.2倍;肿瘤患者输注CD3AK细胞一疗程后,其外周血T淋巴细胞亚群绝对计数有明显升高:总T升高66.0%,Th升高68.0%,Ts升高58.0%;其PBMC的NK杀伤活性由63.0%升高到81.0%,平均升高28.0%.结论:1)脐血单个核细胞是LAK细胞和CD3AK细胞良好的前体细胞.2)LAK和CD3AK细胞的数量和杀伤能力在培养的11天时达高峰,而且CD3AK细胞数量和杀伤活性明显优于LAK细胞.3)CD3AK细胞输注能明显提高肿瘤患者外周血T淋巴细胞亚群绝对计数和PBMC的NK活性.4)肿瘤患者的外周血T淋巴细胞计数和PBMC的NK活性测定可望成为肿瘤生物治疗的一个近期疗效参数.     

B-NHL患者NK细胞中CD16ζ表达及利妥昔单抗与LAK细胞的联合抗瘤作用

背景与目的:自然杀伤细胞(nature killer cell,NK)是抗体依赖细胞介导的细胞毒作用的主要效应细胞,肿瘤患者普遍存在NK细胞活化功能的缺陷可能会影响单克隆抗体的治疗效果.因此如能逆转NK细胞的CD16ζ链信号转导的功能缺陷,并与单克隆抗体联合免疫治疗,可能会产生协同抗肿瘤作用.本研究的目的是了解B细胞非霍奇金淋巴瘤(B-cell non-Hodgkin's lymphoma,B-NHL)患者是否存在NK细胞的活化障碍,体外白细胞介素-2(interleukin-2,IL-2)是否能完全逆转其活化障碍,并观察利妥昔单抗与LAK细胞联合对肿瘤细胞的杀伤作用.方法:使用密度梯度离心方法分别分离69例B-NHL患者和30例健康志愿者外周血单个核细胞(peripheral blood mononuclear cell,PBMC),将两种PBMC在体外与1 000 U/ml IL-12共同培养制备LAK细胞,流式细胞仪检测PBMC和LAK细胞中CD16ζ链的阳性率和平均荧光强度.流式细胞仪检测Raji细胞表面CD20的表达;Annexin V/PI方法检测利妥昔单抗单药对Raji细胞的促凋亡作用,乳酸脱氢酶(lactate dehydrogenase,LDH)释放实验进行杀伤活性的检测.结果:在B-NHL组和健康对照组,CD56 细胞表达CD16ζ链的阳性率为(63.3±16.4)%、(97.8±3.1)%(P＜0.001),CD16ζ链MFI值分别为1.3±1.3和3.6±1.7(P＜0.001).在体外1 000 U/ml的IL-2共培养的LAK细胞中,两组CD16ζ链的阳性率分别为(99.3±4.1)%和(99.7 3.9)%,其MFI值分别为29.2±12.5和31.4±13.8,均无显著性差异(P=0.15和P=0.44).40 μg/ml利妥昔单抗可以完全结合细胞表面CD20抗原,在24 h时才开始出现对Raji细胞的明显的凋亡作用.利妥昔单抗与LAK细胞联合对Raji细胞的杀伤率在不同的浓度组均明显高于不加利妥昔单抗组(P＜0.05).LAK细胞与Herceptin(40 μg/ml)联合的杀伤率与不加Herceptin组相比,在各效靶比浓度梯度均无明显提高(P＞0.05).LAK细胞与利妥昔单抗联合对Jurket细胞的杀伤率在各效靶比浓度梯度均与不加利妥昔单抗组无显著性差异(P＞0.05).结论:B-NHL患者普遍存在NK细胞CD16ζ链的表达下调,高剂量的IL-2可以显著增强CD16ζ链的表达,利妥昔单抗与LAK细胞联合可增强对Raji细胞的抗肿瘤作用.     

Glycoproteic nature of surface molecules of effector cells with lymphokine-activated killer (LAK) activity. Evidence that T11, T8 or T3 molecules are not involved in tumor-cell lysis by LAK effector T cells

Human peripheral blood mononuclear cells cultured in the presence of interleukin-2 (IL-2) acquire the capability of lysing NK-resistant fresh tumor target cells. In an attempt to delineate the surface structure(s) present on the effector cells, the latter were first treated with different amounts of pronase and neuraminidase. The effect of the enzymes on cytolytic activity against fresh melanoma cells was evaluated and compared with the NK-like activity against K562 target cells of the same effector population. At a pronase concentration of 0.01 mg/ml, no inhibition of NK-like activity was detected, whereas LAK activity was inhibited by more than 75%. In addition, neuraminidase had no effect on NK-like activity, even at 1 U/ml, whereas as little as 0.03 U/ml inhibited LAK activity by more than 75%. Metabolic inhibition of N-linked glycosylation with Tunicamycin prevented the generation of LAK activity, even when added late (18 hr before termination of the culture). Tunicamycin, on the other hand, had no effect on the boost of NK activity induced by IL-2. Provided that LAK activity can also be generated in T-cell (E-rosetting) populations, in the presence of adherent cells, we analyzed the inhibitory activity of monoclonal antibodies (MAbs) to T11, T3 and T8 molecules. While all these MAbs strongly inhibited the specific target cell lysis by alloreactive CTLs, they had no effect on the LAK activity.     

Local anergy rather than systemic anti-tumour immunity to explain tumour growth in an animal model of oral squamous cell carcinoma

A chemically induced syngeneic hamster tumour model of human oral squamous cell carcinoma (OSCC) was used to investigate the possible influence of locally transplanted growing rumours on the immune system of the recipient. Cell activation and cell cytotoxicity assays were performed in vitro using the colorimetric MTT assay to measure any possible changes. The fast growing nature of the tumour model if grafted locally as a fragment was confirmed but not if injected as a single cell suspension (SCS). Stimulation (Concanavalin A) of spleen cells from normal and from tumour bearing animals showed that there was a minor though statistically significant decrease in the mitogenic response of the latter. Thus, the respective stimulatory indices (SI) were 4.06+/-1.61 and 2.06+/-0.87 (0.02

0.01). No significant difference was observed when spleen cells were stimulated with interleukin-2 (IL-2), although there was a similar trend. Pre-immunisation of animals with irradiated autologous SCS three weeks prior to grafting, resulted in a significant decrease in the tumour growth rate of subsequently grafted tumour. Thus, the mean If: SD (weight of takes in mg) for the successful takes of untreated (n=10) and treated (n=9) groups were 52.0+/-52.2 and 25.7+/-19.4 (0.02

0.05) respectively. The number of cases with no tumour takes were 2 of 10 (20%) and 6 of 9 (66%) respectively. In a separate experiment groups of 5 animals were immunized with an increasing number of cells as irradiated SCS, the results of which demonstrated an inverse correlation between the rate of tumour growth and the number of injected tumour cells. The addition of irradiated SCS to IL-2 activated normal spleen cells (LAK cells) in vitro led to a dose-related decrease in the efficiency of cytotoxicity of latter when tested against an xenogeneic super-sensitive surrogate tumour target cell line (Fen cells). Thus, the percent killing by IL-2-activated normal spleen cells was 56.4%. The corresponding mean values for IL-2 activated normal spleen cells in the presence of tumour SCS at 25/1 and 50/1 ratios were 35.9% (p<0.05) and 11.9% (p<0.001) respectively. Ln an attempt to establish the presence of T suppressor cells, spleen cells from tumour bearing animals were injected concomitantly with SCS into 5 recipients. After four weeks no tumour growth had occurred. In conclusion we demonstrated that the presence of injected or grafted tumour had only a minor effect on systemic immune function but induced a strong local anergic effect. This local anergic effect was demonstrable as blocking of LAK activity and thus perhaps allowed suppression of the functional activities of incoming immunocompetent cells.     

Role of fibrin coagulation in protection of murine tumor cells from destruction by cytotoxic cells

We have previously proposed that fibrin deposition on tumor cells during their migration in the blood could protect them from elimination by natural killer (NK) or other cytotoxic cells. Anticoagulant drugs could prevent fibrin coagulation and increase the efficiency of cytotoxic effector cells in tumor cell elimination. To further investigate the protective roles of fibrin, we studied in vitro the susceptibility of various murine tumor cells to the cytotoxic activity of NK or lymphokine activated killer (LAK) cells in the presence of murine plasma or serum. In the first set of experiments, tumor cells were incubated with plasma (at dilutions of 1:20-1:160) for 30 min before effector cells were added. Similarly, effector cells were first incubated with plasma before mixing with radiolabeled target cells for cytotoxicity assay. In some experiments target and effector cells and plasma were mixed simultaneously. The cytotoxic activity of both NK and LAK cells was inhibited if coagulation occurred around tumor-target or effector cells. Tumor cells were also protected when both target and effector cells were simultaneously mixed and trapped in the fibrin clot. Inhibition of the cytotoxic activity of effector cells against tumor cells was positively correlated with the level of fibrin clot formation. When the larger clot was formed and more radiolabeled tumor cells were trapped in the clot, the higher level of inhibition of cytotoxicity was observed. In contrast, serum did not affect the cytotoxic activity of NK or LAK cells. To exclude possible non-coagulation-related effects of plasma on LAK cells, a cytotoxicity series of experiments was performed using purified fibrinogen and thrombin. When fibrinogen and thrombin were preincubated with tumor cells or LAK cells or all components were admixed simultaneously, substantial protection of tumor cells from destruction by LAK cells was also observed. However, when heparin was added, fibrin coagulation was prevented and cytotoxic activity of LAK cells was restored. Inhibition of LAK cytotoxicity and protection of tumor cells by fibrin coagulation were mostly due to the prevention of tumor-effector cell conjugate formation. Adding plasma at postbinding time periods (15-30 min after mixing effector and target cells) did not affect the ability of LAK cells to kill tumor cells confirming that fibrin coagulation influenced the binding rather than the lytic phase of cytotoxic cell activity.     

加热处理后人黑色素瘤A375细胞对NK和DC细胞免疫活性的影响

目的： 研究经加热处理的人黑色素瘤细胞A375对外周血单个核细胞来源的自然杀伤(NK)细胞及树突状细胞(DC)免疫活性的影响并探讨其作用机制。方法：体外培养人外周血单个核细胞来源的NK和DC细胞,并使其与43 ℃水浴加热后的A375细胞共孵育24 h。应用CCK-8法测定加热和非加热组效靶比(NK∶DC∶A375)分别为1∶2∶1、3∶6∶1、6∶12∶1时NK/DC细胞的杀伤率;应用酶联免疫吸附法(ELISA)检测上述各效靶比组NK细胞培养液中γ-干扰素(γ-INF)的释放情况。结果：在每个效靶比,与非加热组比较,加热组NK/DC的杀伤率均明显提高(P<0.05),且其杀伤率随NK/DC细胞的浓度升高而升高(P<0.05)。在加热组和非加热组,含有DC较不含DC组杀伤率均明显提高(P<0.05)。加热组NK细胞γ-INF释放均较非加热组明显增加(P<0.05)。结论：经加热处理后的黑色素瘤细胞A375能够刺激NK细胞分泌更多的γ-INF,明显提高NK细胞的杀伤活性,且DC在其中起着重要作用。     

Reversible inhibition of lymphokine-activated killer cell activity by lipoxygenase-pathway inhibitors

Natural killer (NK) cells and lymphokine-activated killer (LAK) cells are anomalous cytotoxic cells which are potentially important in host defense against cancer. Several studies have demonstrated that natural killer (NK) cell activity can be suppressed by chemical inhibitors of the lipoxygenase pathway through inhibition of the production of leukotriene B4 (LTB4). The present study investigated the effects of the lipoxygenase inhibitors BW755C and nordihydroguaiaretic acid (NDGA) on NK and LAK cell activity. NK cell function of fresh peripheral blood mononuclear cells (PBMC) was determined via a standard chromium release assay employing K562 as the tumor target. The LAK cell activity of PBMC which had been stimulated with 10 IU of interleukin-2 for 72 hr was determined against the NK-resistant cell line Daudi. Both BW755C and NDGA inhibited NK and LAK cell function at a variety of concentrations. Indomethacin, a prostaglandin synthesis inhibitor, did not bring about an appreciable diminution in NK or LAK cell activity. Inhibition of NK and LAK cell activities by BW755C and NDGA could be reversed by washing the effector cell suspensions prior to the cytotoxic assay or by adding LTB4 (10(-11)-10(-8) M) directly to the effector:target suspensions. These data indicate that certain arachidonic acid oxidation products of the lipoxygenase pathway are essential for the function of LAK cells.     

Relation of natural killer cell line NK-92-mediated cytolysis (NK-92-lysis) with the surface markers of major histocompatibility complex class I antigens, adhesion molecules, and Fas of target cells.

Natural killer (NK) cell line NK-92 has recently been established by Klingemann et al. In this study, we compared the NK-92-mediated cytolysis (NK-92-lysis) with the killing of healthy volunteers' NK cells and lymphokine-activated killer (LAK) cells. The NK-92-lysis was partially different from the NK- and LAK-lysis. 1) The NK-92 could kill most of major histocompatibility complex (MHC) class I antigen-positive tumor cells. 2) The NK cells killed a myeloid leukemia cell line K562, but the NK-92 showed low killer activity against it. 3) The LAK cells could not kill a CD58-deficient cell line OKM-2T, whereas the NK-92 could kill it sufficiently. 4) The NK-92 could not kill CD54-, CD102-deficient cell lines T98G and U373MG; however, the LAK cells could kill them. Blocking tests using specific antibodies revealed the reason for these differences. The K562 expressed relatively low levels of CD54 and CD102. When the K562 was pretreated with anti-CD54 and anti-CD102, the NK-92 could not kill it at all, whereas the NK cells could still kill it, although the killing level decreased. The NK-92 could not kill the anti-CD54- and anti-CD102-treated OKM-2T. The LAK cells could not kill anti-CD58-treated U373MG and T98G. These findings suggest that NK-92-lysis may require the CD54 and CD102 but that NK-lysis does not require them as much, whereas the LAK-lysis may be rather in relation with the CD58. The NK-92 has high killer activity, and may be applicable for clinical use. However, it should be considered that the NK-92 cannnot kill CD54-, CD102-deficient tumor cells.     

Lack of MHC class I surface expression on neoplastic cells and poor activation of the secretory pathway of cytotoxic cells in oral squamous cell carcinomas.

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells use the secretory pathway of perforin/granzymes to kill their target cells. In contrast to NK cells, CTL responses are MHC class I restricted. In this study we analysed the relative activation of CTL and NK cells in relation with MHC class I expression on oral squamous cell carcinomas (OSCCs). MHC class I expression was investigated in 47 OSCCs by immunohistochemistry using HCA2, HC10 and beta2-m antibodies. The presence of CTLs, NK cells, and its activation, was investigated in 21 of these OSCCs using respectively, CD8, CD57 and GrB7 antibodies. The Q-Prodit measuring system was used for quantification of cytotoxic cells. All OSCCs showed weak or absent staining of beta2-m on the cell surface. The absence of beta2-m was significantly associated with absent expression of MHC class I heavy chain as detected by HC10 antibody (P = 0.004). In tumour infiltrates CTLs always outnumbered NK cells, as reflected by the ratio CD57/CD8 being always inferior to one (mean: 0.19; SD: 0.15). The proportion of activated cytotoxic cells as detected by granzyme B expression was generally low (mean: 8.6%; SD 8.9). A clear correlation between MHC class I expression and the relative proportion of NK cells/CTLs was not found. This study shows that the majority of OSCCs show weak or absent expression of MHC class I molecules on the cell surface, possibly due to alterations in the normal beta2-m pathway. The low proportion of granzyme B-positive CTLs/NK cells indicates that the secretory pathway of cytotoxicity is poor in these patients. The lack of correlation between MHC class I expression and CTL/NK cell activation as detected by granzyme-B expression suggests that, next to poor antigen presentation, also local factors seem to determine the final outcome of the cytotoxic immune response.     

Synergistic action of a plant rhamnogalacturonan enhancing antitumor cytotoxicity of human natural killer and lymphokine-activated killer cells: chemical specificity of target cell recognition.

The spontaneous natural killer (NK) and lymphokine-activated killer (LAK) cytotoxicity of highly purified CD56+CD3- NK cells (90 to 95%) against NK-sensitive and NK-insensitive target cells was drastically enhanced when a rhamnogalacturonan contained in a commercially available Viscum album extract was present during 4-h cytotoxicity assays. This enhancement correlated strictly with an increased formation of NK cell or LAK cell/tumor cell conjugate formation. Information on the chemical specificity of NK cell and LAK cell interaction with target cells and with the rhamnogalacturonan was obtained from inhibition studies. The most efficient inhibitors (100% inhibition at 5 mg/ml) were acetylated D-mannose and acetylated L-mannonic acid gamma-lactone. They specifically inhibited in a dose-dependent manner: (a) the cytotoxicity of NK cells against K562 cells and the formation of NK cell/K562 cell conjugates; (b) the cytotoxicity of LAK cells against K562 cells and Daudi cells as well as the formation of LAK cell/K562 cell and of LAK cell/Daudi cell conjugates; and (c) the synergistic effects of the rhamnogalacturonan in the cytotoxicity assays and the target cell-conjugate formation assays with NK cells and LAK cells. The inhibitory effects observed after pretreatment of NK cells or LAK cells with acetylated mannose were completely reversible, but that obtained with acetylated mannonic acid gamma-lactone was only partly reversible, and the degree of reversibility depended on the inhibitor concentration applied during pretreatment. Nonacetylated mannose or mannose derivatives up to concentrations of 20 mmol showed no inhibitory effects. A mechanistic model representing the interaction of NK cells and LAK cells with target cells and with rhamnogalacturonan is proposed.     

Decreased susceptibility of lined human gliosarcoma cells to lymphokine-activated killer cell cytolysis by gamma-interferon treatment

The susceptibility of the established cultured gliosarcoma line GI-1 to lymphokine-activated killer (LAK) cells was analyzed with and without interferon (IFN)-gamma treatment of target GI-1 cells. IFN-gamma treatment decreased the susceptibility of GI-1 cells to LAK cell cytolysis in a dose-dependent manner. Acid treatment of GI-1 cells increased their susceptibility to cytolysis compared with untreated cells. IFN-gamma treatment and acid treatment of GI-1 cells respectively increased and decreased the expression of class I HLA antigens on GI-1 cells. The susceptibility of GI-1 cells to LAK cell cytolysis and their expression of HLA class I molecules were inversely correlated. Subpopulation depletion experiments on the LAK cells with monoclonal antibodies and complement revealed that phenotypically natural killer type (CD16+) cells had a high cytotoxic activity against untreated GI-1 cells but a relatively low activity against IFN-gamma-treated GI-1 cells in both the precursor and effector phases. On the other hand, phenotypically T-type (CD3+) cells did not show these tendencies at all in both the precursor and the effector phases.