血清抗体及补体对利妥昔单抗介导的NK 细胞杀伤Raji细胞作用的影响

 【摘要】 目的 体外检测血清抗体及补体对利妥昔单抗介导的NK细胞对Raji细胞杀伤作用的影响。方法 通过巢式反转录聚合酶链反应（nest-PCR）检测NK细胞上FcγRⅢa（CD16a）基因型;流式细胞术检测加入血清抗体前后NK细胞上FcγRⅢa的表达;DIO-PI双荧光染色法检测添加血清补体及抗体后NK细胞对Raji细胞的杀伤效应强度。结果 添加血清补体组杀伤率较未添加组明显增强,而添加血清抗体组杀伤率较未添加组明显减弱,在FcγRⅢa-158V／V组内,添加血清补体组、添加血清抗体组与未添加组细胞毒性指数分别为（94.25±1.79） %、（59.79±0.66） %和（69.05±2.38） %,三组之间两两比较均P＜0.05;在FcγRⅢa-158V／F组内,添加血清补体组、添加血清抗体组与未添加组细胞毒性指数分别为（66.71±5.57） %、（18.13±2.99） %和（39.63±3.86） %,三组之间两两比较均P＜0.05。结论 血清补体可增强利妥昔单抗介导的NK细胞对Raji细胞的杀伤作用。血清抗体会减弱利妥昔单抗介导的NK细胞对Raji细胞的杀伤作用。使用肿瘤特异性单克隆抗体（MAb）治疗肿瘤患者时,同时输注新鲜冷冻血浆可提高其抗肿瘤疗效;而MAb与静脉注射用免疫球收白（IVIG）同时使用则可能降低其抗肿瘤疗效。     

利妥昔单抗对弥漫大B细胞淋巴瘤患者Th17细胞及相关细胞因子影响的体外实验

目的 探讨利妥昔单抗对弥漫大B细胞淋巴瘤(DLBCL)患者Th17细胞及相关细胞因子体外的影响及其意义.方法 DLBCL初治患者(DLBCL组)20例和健康对照者(健康对照组)20名,每个对象分别采集4份外周血标本,分离外周血单个核细胞(PBMC),按照培养条件的不同分成4个亚组:空白组(A亚组)、加入利妥昔单抗组(B亚组)、加入利妥昔单抗和血清组(C亚组)和极化组(D亚组)[加白细胞介素6(IL-6)和转化生长因子(TGF-β)].培养后分别采用流式细胞术检测各亚组外周血PBMC中Th17细胞比例,酶联免疫吸附法(ELISA)检测上述培养液中白细胞介素17(IL-17)的表达水平.结果 健康对照组中,D亚组的Th17细胞比例和IL-17水平分别为(17.12±4.90)％、(45.735±10.012)pg/ml,均高于A、B、C亚组(P＜0.05),A、B、C亚组之间比较差异均无统计学意义(P＞0.05).DLBCL组A亚组的Th17细胞比例及IL-17水平为(0.69±0.24)％、(6.012±1.312)pg/ml,均低于健康对照组A亚组的(2.43±0.61)％、(8.217± 1.681)pg/ml(P＜ 0.05);在与利妥昔单抗一起体外培养后,DLBCL组中B、C和D亚组的Th17细胞比例分别为(2.34±0.48)％、(2.31±0.53)％和(16.92±4.81)％,IL-17水平分别为(7.944±1.538) pg/ml、(7.957±1.533) pg/ml和(44.417±9.881) pg/ml,均高于A亚组,且D亚组明显高于B、C亚组(P＜0.05).B、C亚组之间比较差异无统计学意义(P＞0.05).结论 体外实验证实,DLBCL患者外周血PBMC中Th17细胞比例低于健康人,利妥昔单抗作用于DLBCL患者外周血PBMC后可升高Th17细胞比例.     

抗体依赖细胞介导的细胞毒作用在利妥昔单抗耐药机制中的研究进展

利妥昔单抗是一种在B细胞非霍奇金淋巴瘤（B-NHL）中广泛应用的单抗类靶向药物,作用于CD20抗原.其联合化疗显著改善了B细胞淋巴瘤患者的预后.但是仍有部分患者对利妥昔单抗治疗无效或在治疗有效后短期内复发,提示可能存在利妥昔单抗的耐药.利妥昔单抗对B细胞淋巴瘤细胞的杀伤机制主要包括直接诱导凋亡、抗体依赖细胞介导的细胞毒作用（ADCC）、补体依赖细胞毒作用（CDC）等,不同的作用机制可能存在不同的耐药机制,其中哪一种发挥了主要作用尚不明确.文章对利妥昔单抗ADCC作用机制中可能存在的影响疗效的因素进行综述.     

去甲斑蝥素联合IL-12、IL-15处理增强PBMC对Raja细胞的杀伤效应【创新点评：中药抗癌药物去甲斑蝥素增强PBMC对淋巴瘤的杀伤－郑胡镛】

目的:探讨细胞因子IL-2、IL-15激活的人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)对去甲斑蝥素(norcantharidin,NCTD)处理后的人Burkitt淋巴瘤Raji细胞的杀伤效应及其可能机制.方法:锥虫蓝拒染法检测NCTD对Raji细胞和PBMC细胞增殖的影响;LDH释放法检测IL-2、IL-15激活后的PBMC对K562细胞和Raji细胞的杀伤;流式细胞术(flow cytometry,FCM)检测IL-2、IL-15诱导后PBMC表面NKG2D的表达,以及NCTD作用前后Raji细胞表面NKG2D配体(MICA、MICB、ULBP1、ULBP2、ULBP3)的表达.结果:NCTD抑制Raji细胞的增殖,具有剂量、时间依赖性(P＜0.05),但对PBMC增殖无影响(P＞0.05).在效靶比为10∶1、20∶1时,IL-2、IL-15激活的PBMC对K562细胞的杀伤率较未激活的PBMC明显增高[(52.42±3.89)％vs (15.82±5.12)％,(79.55±9.22)％vs(27.67±3.66)％,P＜0.05];PBMC对NCTD处理后Raji细胞杀伤率较未经NCTD处理的Raji细胞明显增高[(23.63±6.20)％vs (5.04±1.25)％,(41.80±4.09)％ vs (8.59±2.19)％;P＜0.05],且IL-2、IL-15激活的PBMC对NCTD处理后Raji细胞的杀伤率较NCTD处理前明显提高[(38.97±2.76)％ vs(13.19±3.67)％,(63.09 ±7.30)％ vs(19.89 ±4.15)％;P＜0.05].激活后PBMC表面NKG2D表达率升高[(44.91±5.85)％vs (25.28±7.69)％,P＜0.05].NCTD作用后Raji细胞表面NKG2D的配体ULBP2表达显著升高[(12.69±3.99)％vs(1.03 ±0.42)％,P＜0.05],而其他配体MICA、MICB、ULBP1、ULBP3的表达无明显变化(P＞0.05).结论:NCTD联合细胞因子IL-2、IL-15处理能增强PBMC对Raji细胞的杀伤效应,其机制与NCTD上调Raji细胞表面ULBP2表达和细胞因子上调PBMC表面NKG2D表达有关.     

HSP70-TKD诱导的NK细胞杀伤肝癌细胞的实验研究

目的:探讨从人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)中获得足够数量和高细胞毒活性的CD3-CD56 NK细胞的方法,并观察其对肝癌细胞HepG2杀伤效应.方法:以干细胞培养基(SCGM)为基础培养基,在不同浓度的抗CD3单抗、IL-2和PHA的培养条件下,从健康人PBMC中高效扩增获得CD3-CD56 NK细胞;以不同剂量的HSP70-TKD诱导NK细胞的活性,MTT法测定HSP70-TKD诱导的NK细胞对HepG2和榄香烯处理的HepGZ细胞的杀伤活性.结果:建立了从人PBMC体外扩增CD3-CD56 NK细胞体系:以SCGM为基础培养基,在600 U/mL IL-2、500 μg/L抗CD3单抗和50 mg/L PHA联合作用下,培养14 d细胞扩增了116倍,舍有(66.97±8.76)?3-CD56 NK细胞;HSP70-TKD诱导的NK细胞对HepG2杀伤活性低.与未经TKD诱导的NK细胞之间差异无统计学意义,P=0.298;而对税香烯处理过的HepG2杀伤活性高,诱导组与未诱导组之间差异有统计学意义,P=0.008;当TKD浓度为2.0 mg/L时,杀伤活性最高为(58.8±3.4)%.结论:以干细胞生长培养基为基础培养基,在抗CD3单抗和IL-2协同刺激下体外大量扩增NK细胞,HSP70-TKD诱导的NK细胞对细胞膜HSP70阳性的肿瘤细胞杀伤活性高,而对于细胞膜HSP70低表达的肿瘤细胞杀伤活性低.     

CD137单抗联合IL-15体外扩增NK细胞及其对肺癌细胞的杀伤活性

目的:建立CD137单抗联合IL-15体外扩增NK细胞的方案,研究扩增后NK细胞对肺癌细胞的杀伤活性。方法:分离健康人外周血单个核细胞,经免疫磁珠阴性分选获取CD3-CD56+NK细胞,将NK细胞分成对照组、IL-2组、IL-2+CD137单抗组、IL-2+IL-15组、IL-2+CD137单抗+IL-15组进行培养,经锥虫蓝染色法计算NK细胞的扩增倍数,流式细胞术检测NK细胞的表型,LDH酶释放法检测NK细胞对肺癌细胞株A549的杀伤活性,ELISA法检测扩增后NK细胞培养液上清中IFN-γ的分泌量。结果:经磁珠分选后NK细胞纯度为(93.28±3.21)%。IL-2+CD137单抗+IL-15组NK细胞扩增倍数明显高于IL-2+CD137单抗组、IL-2+IL-15组、IL-2组及对照组[(86.20±5.00)vs(60.01±5.00)、(49.06±4.39)、(17.04±1.49)、(3.95+0.23)倍,P<0.01]。IL-2+CD137单抗+IL-15组NK细胞对A549细胞的杀伤效率明显高于其他各组[(93.14±3.27)%vs(83.15±4.03)%、(71.25±3.24)%、(62.27±3.01)%、(49.38±2.35)%,P<0.01]。IL-2+CD137单抗+IL-15组培养液上清中的IFN-γ的分泌水平明显高于IL-2+CD137单抗组、IL-2+IL-15组、IL-2组及对照组[(296.25±9.79)vs(260.47±11.55)、(201.13±6.36)、(138.36±6.09)、(38.42±3.56)pg/ml,P<0.01]。结论:CD137单抗联合IL-15能高效扩增NK细胞,扩增的NK细胞高效杀伤A549肺癌细胞。     

补体依赖细胞毒作用通路与利妥昔单抗耐药

利妥昔单抗是针对B细胞表面标志CD20抗原的单克隆抗体,利妥昔单抗联合化疗显著改善了B细胞淋巴瘤患者的预后。但是仍有部分患者对利妥昔单抗治疗无效或在治疗有效后短期内复发,说明利妥昔单抗联合化疗不足以彻底清除淋巴瘤细胞,提示存在着一定的耐药性肿瘤细胞。补体依赖细胞毒作用(complement-dependent cytotoxicity,CDC)是利妥昔单抗对淋巴瘤细胞的主要杀伤机制之一,CDC作用通路中的任何一个环节异常都有可能影响利妥昔单抗的疗效。CD20抗原是利妥昔单抗发挥作用的基础,CD20抗原的表达强度、其编码基因的多态性以及其在细胞膜上的承载结构———脂质筏均可能影响CDC作用;此外,补体的基因多态性(如C1q、CD11b)、血清补体水平及补体调节蛋白的水平均可能影响着CDC作用通路中的不同环节,从而影响利妥昔单抗作用的发挥。随着利妥昔单抗的广泛应用,对其耐药机制的研究日益深入。本文将对目前关于利妥昔单抗CDC作用通路中可能影响其疗效的主要因素进行综述。     

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.     

弥漫大B细胞淋巴瘤患者淋巴细胞亚群特点及利妥昔单抗对其影响的临床研究

目的 探讨不同分期的弥漫大B细胞淋巴瘤(DLBCL)患者外周血淋巴细胞亚群特点,以及应用利妥昔单抗治疗后淋巴细胞亚群的变化.方法 应用流式细胞术检测初治DLBCL患者应用利妥昔单抗(R)加CHOP方案(35例),CHOP方案(60例)化疗前后以及健康人(33名)外周血的淋巴细胞表型.结果 DLBCL患者总T细胞、CD+4细胞计数,CD4/CD8比值均低于健康对照组,CD+8细胞计数则明显高于健康对照组,Ⅲ～Ⅳ期患者尤为突出(P＜0.01);Ⅰ～Ⅳ期患者NK细胞计数均显著高于健康对照组(P＜0.01),但Ⅲ～Ⅳ期较Ⅰ～Ⅱ期患者有所降低(P＜0.05);Ⅰ～Ⅳ期DLBCL患者外周血B细胞计数与健康对照组差异无统计学意义(P＞0.05).RCHOP组患者与CHOP组患者化疗后总T细胞、CD+4细胞、CD+8细胞、NK细胞计数,CD4/CD8比值差异无统计学意义(P＞0.05),但化疗后前者B细胞计数显著低于后者(P＜0.01).结论 DLBCL患者存在细胞免疫调节异常,并与临床分期、病情进展密切相关.淋巴细胞亚群检测为DLBCL诊断、病情评价提供了简易可行的免疫学指标.利妥昔单抗对患者T细胞及NK细胞无明显影响,可引起B细胞显著降低,但机会性感染发生率并未增加.     

脐血CD_3AK细胞的制备、生物活性测定及临床应用初探

 目的　研究用脐血单个核细胞制备CD3 AK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 +CD3 Ab诱导LAK和CD3AK细胞 ,并测其扩增数量和对K5 6 2细胞的杀伤活性 ;测定肿瘤患者用CD3 AK细胞治疗前后外周血单核细胞(PBMC)的NK杀伤活性。结果　脐血LAK细胞和脐血CD3 AK细胞均于培养后第 11天时扩增倍数最高 ,分别是培养前的 18倍、2 4倍 ,对K5 6 2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CD3 AK细胞一疗程后 ,其PBMC的NK杀伤活性由 6 2 %升高到 82 % ,平均升高 32 %。结论　 (1)脐血单个核细胞是LAK细胞和CD3 AK细胞良好的前体细胞。 (2 )LAK和CD3 AK细胞的数量和杀伤能力在培养的第 11天时达高峰 ,而且CD3 AK细胞数量和杀伤活性明显优于LAK细胞。 (3)CD3 AK细胞输注能明显提高肿瘤患者PBMC的NK活性。 (4 )肿瘤病人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     

Identification of a novel CD56- lymphokine-activated killer cell precursor in cancer patients receiving recombinant interleukin 2.

Circulating lymphokine-activated killer (LAK) cell activity in cancer patients receiving recombinant interleukin 2 (rIL-2) therapy is confined to cells expressing the CD56- surface marker. However, CD56- cells from these patients but not normal individuals have been reported to exhibit LAK cytotoxicity only following in vitro activation with rIL-2. Studies were performed to document the existence of CD56- LAK precursor cells and to phenotypically characterize this population in patients receiving rIL-2 therapy using fluorescence-activated cell sorter-purified CD56- cell subsets. Initial studies confirmed that CD56- cells exhibit NK activity [20 +/- 7 (SE) LU/10(6) cells] but not LAK activity (0 +/- 0 LU/10(6) cells) when evaluated directly from peripheral blood of patients receiving rIL-2. CD56- cells from patients but not normal individuals developed significant LAK cytolytic activity against NK-resistant COLO 205 targets (16 +/- 3 LU/10(6) cells) when cultured for 3 days with 1500 units/ml rIL-2. The CD56- LAK precursor activity was confined to cells expressing a CD56-CD16+ phenotype and a large granular lymphocyte morphology; little or no NK or LAK precursor activity was detectable in CD56-CD5+ T-cells from patients. Phenotypic characterization of CD16+CD56- cells revealed that this population is uniformly CD11a+,CD18+, and CD38+ and is heterogeneous in its expression of CD11b, CD11c, and CD16/Leu 11c. These results indicate that rIL-2 administration induces enhanced LAK precursor activity in a novel population of CD5-CD16+CD56- cells.     

干扰素-γ对乳腺癌细胞Her2/neu表达及131I-Herceptin抑制肿瘤细胞增殖的影响

背景与目的:利用Herceptin对Her2/neu的靶向特性,将放射性核素131I联结到Herceptin进行放免靶向治疗,是治疗转移性乳腺癌的方法之一。而肿瘤摄取标记抗体的量与肿瘤细胞靶位分子的表达量密切相关。本研究应用IFN-γ上调乳腺癌细胞系Her2/neu的表达,以提高131I-Herceptin在乳腺癌细胞系的结合,及131I-Herceptin对乳腺癌细胞增殖的抑制作用。方法:取对数生长期乳腺癌细胞系MCF-7、SKBR-3和BT-474,实验组以终浓度为500U/ml的IFN-γ诱导培养48h,对照组加入不含IFN-γ的等量培养液。诱导前后采用流式细胞仪(FACS)检测3种细胞Her2/neu的表达率及平均荧光强度(MFI)。采用Iodogen法对Herceptin进行131I标记,以高压液相层析法(HPLC)测定131I-Herceptin的放射化学纯度(RCP),以非竞争性饱和结合法测定3种细胞诱导前后131I-Herceptin的结合率(B/T)。采用克隆形成实验评价诱导后131I-Herceptin对3种细胞杀伤效应的变化。实验组与对照组间Her2/neu表达率、MFI、B/T及克隆形成率的差异采用t检验。结果:MCF-7细胞Her2/neu基础表达率为(8.5±1.9)%,诱导后升高至(15.2±2.7)%(t=3.515,P<0.05),MFI从38±7升高至121±17(t=7.823,P<0.01);SKBR-3细胞和BT-474细胞的基础表达率分别为(98.9±1.1)%和(98.1±0.9)%,诱导后分别为(99.7±0.9)%和(99.5±1.2)%,无明显改变(P>0.05),但MFI分别从952±125、1020±98增高至1608±201(t=4.802,P<0.01)和1968±192(t=7.614,P<0.002)。131I-Herceptin在MCF-7、SKBR-3和BT-474的基础结合率分别为(5.2±1.4)%、(35.8±4.5)%和(37.2±3.6)%,诱导后分别升高为(12.3±3.4)%、(48.9±7.1)%和(59.5±8.7)%,对131I-Herceptin的结合倍增比分别为2.4、1.4和1.6倍。实验组3种细胞的克隆形成率分别为(30±4)%、(23±5)%及(19±6%),均显著低于对照组[分别为(49±3)%、(37±6)%、(34±5)%](t=6.574、3.105、3.323,P<0.05)。结论:IFN-γ可以上调乳腺癌细胞系Her-2/neu的表达和肿瘤细胞对131I-Herceptin的结合量,因此也提高了131I-Herceptin对乳腺癌细胞的增殖抑制作用。     

Lymphokine-activated killer cells can kill target cells not only by early killing but also by late killing,and the late killing level against autotumor cell line

Generally, lymphokine-activated killer (LAK) cells kill target cells early after the LAK cells adhere to them. In this study, we describe that LAK cells can also kill at a later time, such as 24-96 h. LAK cells were generated from a cancer patient and healthy volunteers. As target cells, the patient's autotumor cell line H41 was used. When LAK cells were added to the target cells in a culture well, the LAK cells killed the target cells by cell-cell adhesion within 1-4 h (early killing), but not all cells were killed. The LAK cells were then removed. However, the remaining cells ultimately died 24-96 h later (late killing). The late killing was different from the early killing because numerous granules and vacuoles appeared in the cytoplasm. The late killing was not induced by adding supernatant of the LAK cell culture, suggesting that LAK-target cell contact may be necessary for the killing. The cell injury was inhibited by 3-methyladenine (lysosome inhibitor). It suggests that the vacuoles may be caused by activated lysosome. The patient's LAK cells induced late killing at high levels. There was a high percentage of CD8(+)CD16(+) cells in the peripheral blood lymphocytes (PBL). This subset induced late killing more effectively than the CD8(-)CD16(+) subset. Killing was more conspicuous against H41 than against allogeneic cell line T98G. This type of killing is noteworthy for understanding of killing mechanism of LAK cells.     

Preparation and Bioactivity Assay of CD3AK Cell from Lmbilical Cord Blood：—Clinic Report of 10 Tumor Cases

Objective To study the anti-tumor effect of CD3AK cell prepared from umbilical blood,to explore the short-term curative effect on tumor cases and seek better immune index for biotherapy.Methods IL-2 and IL-2 CD3Ab were used to induce LAK cells and CD3AK cells isolated from umbilical blood mononuclear cells(UBMC).The expanding number and bioactivity of LAK cells and CD3AK cells were examined at different time points after culture,the NK activity of peripheral blood mononuclear cells(PBMC)of 10 cases of malignant tumor were determined before and after CD3AK cell adopting immune therapy as well.Results The number and bioactivity(NK killing K562 cell)of LAK and CD3AK cells reached their peaks on 11 th day.The number of LAK and CD3AK cells were 18 folds and 24 folds of that before culture;The NK activities of LAK and CD3AK against K562 were 2.6 folds and 3.2 folds of those before culture respectively.The nK activity for killing K562 cells of malignant tumor patient‘s PBMC was increased from 63%-81% by CD3AK cell transfusing,rising mean 28%.Conclusion (1)The UBMC is a potential and better source of predecessor for LAK and CD3AK;(2)The NK activity of LAK and CD3AK cells from UBMC reached their peaks at 11 th day after culture,and the NK aftivity of CD3AK cells in much greater than that of LAK cells;(3)The NK activity of malignant tumor patient‘s PBMC can be obviously elevated by transfusing CD3AK cell(4)The test of NK activity of PBMC of malignant tumor patient may become an objective immune index for tumor biotherapy.     

单价抗CD20抗体诱导人B细胞淋巴瘤Raji细胞的凋亡

背景与目的:抗CD20抗体和片段已应用于非霍奇金淋巴瘤的临床治疗,但仍需要开发新的抗CD20抗体和片段(未修饰的或放射性标记的),以治疗对美罗华(利妥昔单抗)无反应的患者。鼠源性抗CD20抗体HI47的嵌合抗体片段Fab和F(ab)'2已被构建。本研究目的是观察HI47(鼠抗-CD20抗体)和其嵌合抗CD20抗体片段抑制肿瘤细胞生长和诱导肿瘤细胞凋亡的作用。方法:用免疫荧光法测定抗CD20抗体与CD20+人B细胞淋巴瘤Raji细胞的结合能力;MTT法测定抗CD20抗体片段对Raji细胞生长的影响;用膜联蛋白Ⅴ染色和DNA琼脂糖凝胶电泳检测和验证抗CD20抗体片段诱导Raji细胞凋亡。结果:HI47和其嵌合的抗CD20抗体片段均可与CD20+Raji细胞结合,结合率可达90%以上;HI47不能与美罗华竞争结合Raji细胞;HI47和其嵌合的抗CD20抗体片段浓度为100μg/ml对Raji细胞的抑制率分别为:(57.0±1.5)%、(65.2±2.5)%、(77.2±3.2)%;单价的抗CD20抗体片段Fab(20μg/ml)能够诱导Raji细胞的凋亡,早期凋亡率为17%。结论:HI47的嵌合抗体片段对Raji细胞有抑制作用,能诱导Raji细胞的凋亡。     

Mechanisms of selective killing of neuroblastoma cells by natural killer cells and lymphokine activated killer cells. Potential for residual disease eradication.

Widely disseminated neuroblastoma in children older than infancy remains a very poor prognosis disease. Even the introduction of marrow ablative chemotherapy with autologous rescue has not significantly improved the outlook for these children, presumably because of a failure to eradicate minimal residual disease. One additional approach which may hold promise is the use of immunomodulation with cytokines such as IL2 in the setting of minimal residual disease (MDR), for example after intensive chemotherapy and ABMT. However, considerable variability in the susceptibility of neuroblastoma cells to natural killer (NK) and lymphokine-activated (LAK) killing has been observed, and it is presently unclear how NK and LAK cells recognise neuroblastoma cells. In this paper we examine expression of cell adhesion molecules on neuroblastoma to determine which of these modify interaction with NK and LAK cells. We find that LFA-3 (CD58), the ligand for CD2 is of predominant importance in predicting susceptibility of neuroblastoma to the cytotoxic actions of NK and LAK cells, while expression of ICAM-1 (CD54) may also modify susceptibility. These findings were confirmed by blocking experiments in which co-culture of target cells with ICAM-1 and LFA-3 reduced LAK and NK cytotoxicity. Study of the immunophenotypic features of each patient''s neuroblastoma cells before induction of MRD may be valuable in determining the likely effect of IL2 in predicting disease reactivation.     

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.