PLA表位肽免疫微球诱导CTL对肝癌细胞的杀伤作用

目的〖HT5"SS〗：考察两种载肽聚乳酸（(polylatic acid, PLA)免疫微球M1（hAFP158~166）和M2（hAFP218~226）在体外诱导特异性CTL的能力及其对肝癌细胞的杀伤作用。〖HT5W〗方法： 〖HT5”SS〗制备分别荷载表位肽hAFP158~166、、hAFP218~226的PLA免疫微球M1和M2,体外刺激HLAA2+健康志愿者的外周血单个核细胞（PBMC）作为效应细胞,实验分为3组：对照组、hAFP表达组和hAFP阴性组。采用标准51Cr释放法检测CTL杀伤活性。〖HT5W〗结果：〖HT5"SS〗两种免疫微球在体外均能刺激人PBMC增殖,形成大量可见克隆;两者诱导的效应细胞对hAFP＋的荷肽T2细胞、HepG2和Alexander的杀伤率均达75％以上,均显著高于不表达hAFP的膀胱癌细胞BTT和未荷肽的T2细胞,差异非常显著（P<0.01）,而两者杀伤活性之间没有明显差异（P>0.05）。〖HT5W〗结论：〖HT5"SS〗两种载肽聚乳酸免疫微球均能在体外诱导产生特异性CTL,并对表达靶抗原的肝癌细胞有较强杀伤作用。     

腺病毒介导的HBsAg基因修饰树突状细胞的体外生物学特性

目的： 〖HT5"SS〗探讨腺病毒介导乙型肝炎病毒表面抗原（AdVHBsAg）基因修饰树突状细胞（dendritic cell,DC）瘤苗体外生物学活性。〖HT5W〗方法〖HT5"SS〗：将腺病毒表达载体AdVHBsAg转染人单个核细胞来源的DC,构建AdVHBsAgDC肝癌瘤苗,采用Western blotting法鉴定转染基因表达,FACS检测表面分子和内吞功能,3HTdR法检测T细胞增殖反应的能力,MTT法检测CTL活性。〖HT5W〗结果〖HT5"SS〗：HBsAg基因转染后,Western blotting法检测结果示HBsAg基因表达于转染的DC,表明腺病毒介导的HBsAg基因转染的有效性。AdVHBsAgDC可高表达CD1a、CD11c、 CD83、CD86和HLADR,但内吞功能较DC组降低(P<0.05)。AdVHBsAg DC刺激自体T细胞增殖功能均明显高于DC对照组和AdVLacZDC组(P<0.05)。AdVHBsAg DC体外诱导CTL对HepG2215肿瘤细胞的杀伤作用具有特异性。〖HT5W〗结论〖HT5"SS〗：肝癌相关基因HBsAg可作为乙型肝炎病毒相关性肝癌的切入点,该研究为HBV相关肝癌DC体内免疫治疗提供了实验依据。     

肺癌患者化疗前后免疫细胞格局的改变及其临床意义

目的〖HT5"SS〗：研究肺癌患者化疗前后免疫细胞数量、亚群比例、细胞表型及功能的改变,并探讨其临床意义。〖HT5W〗方法：〖HT5"SS〗 对23例次肺癌患者化疗前后的白细胞、淋巴细胞和中性粒细胞计数,以流式细胞术对CD3+、CD4+及CD8+T细胞亚群比例以及记忆样表型T细胞进行检测和分析;体外以PHA刺激外周血淋巴细胞,培养48 h后检测对K562靶细胞的杀伤效应。〖HT5W〗结果： 〖HT5"SS〗化疗后外周淋巴细胞数量迅速减少,1周左右达最低水平,此后逐渐恢复至化疗前水平;淋巴细胞恢复过程中, CD3+、CD8+T细胞亚群比例升高,记忆样表型T细胞（CD44high,CD62Llow）比例增加;增生期淋巴细胞在体外以PHA刺激后对K562细胞的杀伤能力没有下降,个别患者的杀伤能力还有所增强。〖HT5W〗结论： 〖HT5"SS〗肺癌患者化疗后不仅没有降低免疫功能,反而可增强其免疫效应,该结果为临床肿瘤患者化疗后开展免疫生物治疗提供了实验依据。     

NT4-p53(N15)-Ant融合基因重组腺病毒的构建与鉴定

目的：〖HT5"SS〗构建编码融合基因NT4p53(N15)Ant的重组腺病毒表达载体,为进一步基因治疗的实验研究奠定基础。〖HT5W〗方法〖HT5"SS〗：利用PCR体系延伸互为模板的引物,通过T载体克隆法获得p53(N15)Ant基因克隆,酶切后连入pBV220/NT4质粒,再将融合基因NT4p53(N15)Ant亚克隆至腺病毒的穿梭质粒内,与辅助质粒PJM17共同转染HEK293细胞,通过同源重组获得重组腺病毒Ad.NT4p53(N15)Ant,收集病毒上清,大量扩增并测定其滴度,用RTPCR检测目的基因在293细胞的表达情况。MTT比色法观察重组病毒对HepG2细胞存活率的影响。〖HT5W〗结果：〖HT5"SS〗克隆出p53(N15)Ant基因,经酶切及测序证实结果正确;得到高滴度的（1×1011pfu/ml）重组腺病毒表达载体;反转录聚合酶链反应证实感染Ad.NT4p53(N15)Ant的293细胞中有目的基因的表达。Ad.NT4p53(N15)Ant对HepG2细胞有强烈的杀伤作用,与Ad.GFP比较,NT4 p53(N15)Ant重组腺病毒处理组HepG2细胞的存活率明显降低。〖HT5W〗结论：〖HT5"SS〗通过分子克隆体外重组技术成功制备了NT4p53(N15)Ant复制缺陷型重组腺病毒,为下一步的肿瘤基因治疗奠定了基础。     

Tat蛋白转导结构域介导HSV1-TK导入肝癌细胞及其效应

目的： 〖HT5"SS〗探讨HIV1编码的反式激活蛋白Tat与HSV1TK融合表达对肝癌细胞的杀伤效果。〖HT5W〗方法：〖HT5"SS〗 合成编码HIVTat47~57（Tat 11）的2条寡核苷酸单链,两端分别引入BamHⅠ和Hind Ⅲ 两个酶切位点,退火形成寡核苷酸双链,15%非变性聚丙烯酰胺凝胶电泳判断退火效果;以rpAs16Dr为模板,通过PCR扩增HSV1TK基因,定向克隆至原核表达载体pET32中。将含pET32cTat 11TK的BL21菌通过IPTG诱导表达,表达产物用Ni2+螯合柱亲和纯化,免疫组化分析重组蛋白Tat 11TK的膜结合特性,蛋白印迹技术分析该融合蛋白的穿膜能力,检测和分析TK/GCV、Tat 11TK/GCV对肝癌细胞株HepG2的杀伤作用。 〖HT5W〗结果〖HT5"SS〗： 表达产物SDSPAGE在相对分子质量60 700左右显示条带,符合Tat 11TK与表达标签融合蛋白的理论值,并证明以包涵体的形式表达;通过Ni2+螯合柱亲和纯化获得的Tat 11TK重组融合蛋白,经免疫组化证实能结合到肝癌细胞表面,蛋白印迹结果说明Tat 11TK能有效穿过细胞膜进入HepG2细胞内,同时应用低浓度前药更昔洛韦（GCV,150 μmol/L）能有效抑制HepG2细胞生长。〖HT5W〗结论〖HT5"SS〗： Tat 11TK在原核系统获得高效表达,具有较强的穿膜能力和前药酶活性。     

RNA干扰对Aurora-A在人卵巢癌细胞中表达及细胞增殖的影响

目的： 〖HT5"SS〗探讨应用RNA干扰技术沉默AuroraA基因表达,研究其对人卵巢癌SKOV3细胞增殖的抑制作用。〖HT5W〗方法〖HT5"SS〗： 设计合成两对特异性针对AuroraA基因的Oligo siRNA,转染至SKOV3细胞中,采用RTPCR和Western blot检测AuroraA mRNA和蛋白表达情况,同时利用MTT试验和流式细胞仪观察转染后细胞增殖抑制和凋亡情况。〖HT5W〗结果：〖HT5"SS〗转染Oligo siRNA后,SKOV3细胞AuroraA mRNA表达受抑制(P<0.01),蛋白表达水平降低;细胞增殖的抑制率和细胞凋亡率明显增高(P<005, P<0.01)。〖HT5W〗结论： 〖HT5"SS〗体外合成的特异性针对AuroraA基因的Oligo siRNA对卵巢癌细胞株SKOV3中AuroraA基因表达和细胞增殖均有明显抑制作用,为进一步研究Auroraa基因的功能提供了实验基础。     

脐带源间充质干细胞对异源性脐带血T淋巴细胞激活与增殖的抑制作用

目的：〖HT5"SS〗观察人脐带源间充质干细胞（hUCMSC）对异源性脐带血T淋巴细胞激活与增殖的影响,探讨其在免疫调控中的作用。〖HT5W〗方法： 〖HT5"SS〗建立分离、扩增hUCMSC的方法,检测其MHC表型;实验分3组：(1)单纯脐带血组（阴性组）;（2)脐带血+丝裂原刺激组（对照组）;（3）脐带血+丝裂原刺激 +MSC共培养组（实验组）;流式细胞术检测各组脐带血T细胞及其CD4＋和CD8＋亚型细胞共培养8 h后早期激活标志CD69的表达, MTT法检测各组脐带血T细胞共培养5 d后增殖情况。 〖HT5W〗结果： 〖HT5"SS〗成功建立分离、扩增hUCMSC的方法,免疫表型分析显示hUCMSC不表达HLAⅡ抗原,低表达HLAI抗原;流式细胞术检测示在hUCMSC共培养情况下,经丝裂原刺激后,脐带血T淋巴细胞CD69表达与对照组\[(22. 6±5.2)%\]的阳性率相比较,下降至(7.8±3.5)%（P<0.01）,而且这种抑制对不同亚型T细胞（CD4+/CD8+）均起作用;MTT检测显示在hUCMSC共培养条件下,丝裂原刺激T细胞增殖受到抑制,抑制的程度与hUCMSC的剂量呈依赖性。 〖HT5W〗结论： 〖HT5"SS〗hUCMSC对异源性脐带血T淋巴细胞激活和增殖具有抑制作用,而且这种作用为非选择性的,呈剂量依赖性。     

突变减毒志贺样毒素Ⅰ真核表达载体的构建及其抗卵巢癌的活性

目的： 〖HT5"SS〗构建表达突变减毒的志贺样毒素Ⅰ(Shigalike toxin 1, Stx1) 的真核表达载体,并考察其抗人卵巢癌细胞SKOV3的活性。〖HT5W〗方法〖HT5"SS〗： 重叠PCR法构建毒性为原毒素毒性1/10和1/100的突变减毒Stx1编码序列,TA克隆并测序后分别连入真核表达载体pcDNA3.1。转染SKOV3细胞,RTPCR法检测Stx1 mRNA在SKOV3细胞中的表达,观察突变减毒Stx1对SKOV3细胞周期的影响和致SKOV3细胞死亡的方式。使用SKOV3荷瘤裸鼠模型,不同毒性突变减毒Stx1真核表达载体经脂质体包裹后肿瘤局部注射给药,评价其体内抑瘤能力。〖HT5W〗结果〖HT5"SS〗: 突变减毒Stx1真核表达载体经酶切和测序鉴定与预期突变序列一致;RTPCR证实转染后的SKOV3细胞中存在目的mRNA;体外实验观察到该载体转染可以使人卵巢癌SKOV3细胞的细胞周期阻滞于G2/M期,转染后SKOV3细胞死亡的主要途径是坏死;体内实验显示突变减毒Stx1真核表达载体可以抑制裸鼠体内SKOV3移植瘤的生长。〖HT5W〗结论〖HT5"SS〗: 通过重叠PCR方法获得2种突变减毒Stx1编码序列,成功构建了相应的真核表达载体,并证实该载体具有明显的抗卵巢癌活性。     

丁酸钠诱导HT 29结肠癌细胞凋亡及其对 p 53靶基因的调控

目的：〖HT5"SS〗分析丁酸钠对HT29结肠癌细胞p53三个主要靶基因（p21waf1,bax和gadd45）的调控,并探讨其作用机制。〖HT5W〗方法：〖HT5"SS〗HT29细胞常规培养在含有和不含有丁酸钠的培养液中。分别用MTT和流式细胞仪(flow cytometry,FCM) 检测细胞增殖和细胞周期分布,通过形态学观察、亚G1峰的检测和AnnexinVFITC 双标记观察细胞凋亡情况;RTPCR和Western blot分别检测丁酸钠对p21waf1,bax和gadd45三种基因mRNA和蛋白表达水平的影响。〖HT5W〗结果：〖HT5”SS〗丁酸钠以剂量和时间依赖的方式抑制HT29细胞增殖和诱导凋亡,并阻滞细胞于G1期。RTPCR 和Western blot结果显示丁酸钠可以促进p21waf1和bax基因mRNA和蛋白的表达,而对gadd45基因的表达无明显影响。〖HT5W〗结论：〖HT5”SS〗2.5 mmol/L以上浓度的丁酸钠可以抑制HT29细胞增殖并诱导凋亡,该作用可能通过上调p21waf1和bax基因表达而实现。     

低氧环境下罗勒多糖对乳腺癌细胞TIMPs mRNA表达的影响

目的： 〖HT5"SS〗探讨低氧对人乳腺癌细胞株MDA231金属蛋白酶组织抑制剂（TIMPs）mRNA表达的影响,以及罗勒多糖(basil polysaccharide,BP)对TIMPs的作用。〖HT5W〗方法：〖HT5"SS〗 分别置MDA231细胞株于常氧（21% O2、5% CO2）、低氧（1% O2、5% CO2和94% N2）环境中和罗勒多糖（200 μg/ml）培养6 h,RTPCR技术检测不同处理组细胞TIMP1、2、3 mRNA水平。〖HT5W〗结果： 〖HT5"SS〗MDA231细胞株表达TIMP1、2 mRNA,未检测到TIMP3 mRNA;低氧环境下MDA231细胞株TIMP1、2 mRNA水平显著上升(P<0.05);罗勒多糖处理后TIMPs mRNA的含量,无论常氧组还是低氧组都有显著改变：常氧环境下罗勒多糖可明显下调MDA231细胞株中TIMP1、2 mRNA的表达(P<0.05),而低氧环境下则显著上调其表达(P<0.05)。〖HT5W〗结论：〖HT5"SS〗 罗勒多糖在常氧及低氧环境下对MDA231细胞TIMP1、2基因表达的影响效应截然相反,提示抗肿瘤药物及生物疗法的体外机制研究设计应充分考虑氧环境的影响。     

Perforin-mediated lysis of tumor cells by Mycobacterium bovis Bacillus Calmette-Guérin-activated killer cells.

Immunotherapy with Bacillus Calmette-Guérin (BCG) is clinically established in the treatment of superficial bladder cancer. In our attempt to clarify the underlying immunological mechanism, we could previously show that stimulation of PBMC with BCG leads to the generation of cytotoxic BCG-activated killer (BAK) cells. Among others, these BAK cells as well as lymphokine-activated killer (LAK) cells have been suggested as possible effector cells during BCG therapy. To understand BCG-induced activation of effector lymphocytes more precisely, we investigated the lytic pathways of human BAK cells and compared BAK cell cytotoxicity with LAK cell cytotoxicity. Perforin and Fas ligand (FasL) are the major cytolytic molecules of cytotoxic lymphocytes. Our results demonstrate that BAK and LAK cells showed an increased expression of perforin and FasL as compared with unstimulated controls. Killing of T-24 bladder tumor as well as Jurkat cells by BAK and LAK cells was predominantly mediated via perforin as demonstrated by a drastically reduced lysis in the presence of concanamycin A and EGTA/MgCl2, respectively. In contrast, lysis (radioactive release assay) and membrane disintegration (Annexin V binding) of both targets by BAK and LAK cells could not be blocked with an inhibitory anti-FasL monoclonal antibody (NOK-1). Nevertheless, T-24 and Jurkat were susceptible to killing by recombinant soluble FasL and by Chinese hamster ovary cells expressing membrane-bound FasL. We conclude that cellular mediators of BCG effector mechanisms, such as BAK and LAK cells, kill their targets via perforin and independent of the FasL pathway. Because we also found increased numbers of perforin-expressing lymphocytes in patients after BCG therapy, our findings have potential clinical relevance because BCG therapy would not be impaired by FasL resistance of target cells, which recently has been described for some tumors.     

Induction of intercellular adhesion molecule 1 on small cell lung carcinoma cell lines by gamma-interferon enhances spontaneous and bispecific anti-CD3 x antitumor antibody-directed lymphokine activated killer cell cytotoxicity.

The interaction between LFA-1 and its natural ligand, ICAM-1, plays an important role in leukocyte adhesion and signal transduction. LFA-1-mediated T-cell adhesion is generally activated by CD3-mediated signal in association with T-cell receptor-mediated recognition of the antigen/major histocompatibility complex on antigen-presenting cells. In the present study, we compared spontaneous or bispecific antibody (BsAb)-directed LAK cell cytotoxicity against ICAM-1+ or ICAM-1- small cell lung cancer (SCLC) cell lines. gamma-Interferon (IFN-gamma)-induced ICAM-1 expression on ICAM-1- SCLC cell lines, and susceptibility to LAK cells was increased simultaneously. Increased cytolysis of the IFN-gamma-treated SCLC was inhibited by an anti-ICAM-1 monoclonal antibody (mAb). Furthermore, LAK cell cytotoxicity directed by BsAb, which was composed of OKT3 and anti-SCLC mAb, was also increased by the IFN-gamma treatment of SCLC, and this increase was inhibited by an anti-ICAM-1 mAb but not by anti-Class I or anti-CD2 mAb. These results suggest that a prior administration of IFN-gamma would enhance the efficacy of the following specific targeting therapy utilizing BsAb and LAK cells by up-regulating the ICAM-1 expression on tumor target cells. The combinational use of IFN-gamma and anti-CD3 x anti-tumor BsAb might be a promising way of enhancing LAK cell-mediated adoptive immunotherapy in small cell lung cancer patients.     

Adhesion Molecules on Murine Lymphokine-activated Killer Cells Responsible for Target Cell Killing: A Role of CD2

Lymphokine-activated killer (LAK) cells were induced from C57BL/6 mouse spleen cells and the effects of culture time on the expression of cell surface phenotypes and cytotoxic activity of LAK cells were determined. The expression of CD2 remarkably decreased after culture of LAK cells for 30 days, while LFA-1, a principal adhesion molecule in LAK cells, and CD3 were not changed by the culture. LAK cells cultured for 90 days completely lost CD2. In accordance with the decrease of CD2, the cytotoxic activity of LAK cells declined but a certain leven was retained even after the complete loss of CD2. The established LAK cell clones were also strongly positive for the expression of LFA-1 but negative for CD2. When the LAK cell clones were transfected with the CD2 cDNA, they started to express CD2 on their cell surface and to show greater binding ability and stronger cytotoxicity to target tumor cells. These results indicated that CD2 plays a role as an adhesion molecule responsible for target cell killing in murine LAK cells.     

Restored T-cell activation mechanisms in human tumour-infiltrating lymphocytes from melanomas and colorectal carcinomas after exposure to interleukin-2

We investigated the effects of interleukin-2 (IL-2) exposure on T-cell signal transduction molecules and apoptosis markers in tumour-infiltrating lymphocytes (TIL) isolated from 20 melanoma and 16 colorectal carcinoma metastases and expanded in vitro for therapeutic reinfusion. Before IL-2 culture, TIL showed undetectable or very low levels of T-cell receptor (TCR) epsilon chain, p56(lck), Fas ligand (FasL) and Bax expression, while Bcl-2 values were elevated. Cancer cells were characterised by low or absent Fas and Bcl-2 and high Bax expression. Notably, they also expressed FasL. After 41-48 days of IL-2 culture, TCR epsilon chain and p56(lck) expression of TIL rose to median values of approximately 80 and 30% positive cells, respectively (P<0.001), FasL expression was detected in 45% cells from melanomas (P<0.001) and in 3% from colorectal carcinomas (P=0.09), and Bax-positive cells increased from 17.5 to 70% (P=0.005). Moreover, TCR zeta chain-positive cells were significantly increased from baseline (P=0.001), Bcl-2-positive cells dropped from 50 to 1% (P=0.007) and perforin content was high, while Fas expression was not significantly modified by IL-2 culture. In conclusion, our data suggest that the degree of immunosuppression in TIL from melanomas and colorectal carcinomas is very high, and the apoptosis markers' repertoire of cancer cells resembles that of immune-privileged tissue. Interleukin-2 culture appears to restore lymphocyte activation mechanisms, resulting in consistent FasL expression and perforin production.     

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.     

Conjugate Formation by Leukemic Blasts from Acute Myeloid Leukemia with Cytotoxic Lymphocytes

Conjugate formation by AML blasts with fresh peripheral blood lymphocytes (PBL) and lym-phokine activated killer (LAK) effectors was studied by flow cytometry. Leukemic blasts formed very low numbers of conjugates with fresh PBL and were resistant to natural killer (NK) cytotoxicity. When LAK effectors were used a significant increase in conjugate formation was observed, which in the majority of cases was followed by an increased killing. There was a positive correlation between the percentages of conjugates formed by AML blasts with LAK effectors and the susceptibility to lysis. No significant difference in binding activity between the CD3+ and CD56+ LAK subpopulations was found. There was no correlation between the expression of ICAM-1, LFA-3 and Transferrin receptor (CD71) and the conjugate formation. The blocking of CD71 on the control K562 cell line reduced the conjugate formation with LAK effectors but no such effect could be observed with CD71 + AML blasts.     

Target lysis by human LAK cells is critically dependent upon target binding properties, but LFA-1, LFA-3 and ICAM-1 are not the major adhesion ligands on targets.

The cytotoxicity mediated by the CD2+ CD3- lymphocyte subset, either NK or LAK, is puzzling since no specific antigen recognition structures, equivalent to the CD3-associated heterodimer T-cell receptor, have been recognized on these cells so far. The possibility exists that the CD3- cytotoxic effectors recognize their targets through non-specific adhesion mechanisms. The goal of this study was: (a) to examine the correlation between binding properties and susceptibility to lysis of 6 informative target cell lines; (b) to evaluate the role, as ligands on these targets, of adhesion molecules such as LFA-1, LFA-3 and ICAM-1. The effectors used in this study were IL-2-activated LGL, predominantly CD3-, or highly purified CD3- lymphocytes from normal human donors. The 6 target lines studied included 2 pairs of EBV-transformed B-cell lines (721 LCL vs. 721.134, and MM vs. MM-10F2) in which the parental lines were resistant to lysis while HLA variants were susceptible. A third pair was the Daudi Burkitt cell line, susceptible to LAK lysis, and an HLA-positive transfected Daudi line which was more resistant to lysis. The binding properties of these targets to LAK effectors (conjugate formation) were evaluated using a sensitive double fluorescence flow cytometry method. In each pair examined, the susceptible targets formed more conjugates and were surrounded by more cytotoxic LAK effectors than their resistant counterparts, indicating that the conjugation properties of targets are closely correlated with their susceptibility to LAK lysis. The expression of adhesion molecules on the informative targets was examined by indirect immunofluorescence and their role was evaluated by inhibition of lysis after pre-coating the targets with the relevant antibodies. The differences in the expression of the classical cell-cell adhesion molecules LFA-1, LFA-3 and ICAM-1 on the target surfaces were only marginal, insufficient to explain the striking differences in susceptibility to lysis and in binding properties. Coating the target cells with antibodies directed against these adhesion determinants had no effects on the lysis of susceptible target cells. The same antibodies reacting with the LAK effectors did inhibit lysis. Taken together, these results suggest that, on the targets, presently undefined membrane adhesion structures may have a major role in conjugate formation between target and CD3- effectors and determine the susceptibility of the targets to lysis.     

IL-12 and IL-2 potentiate the in vitro tumor-specific activity of peripheral blood cells from cervical cancer patients

We have carried out a detailed analysis of the cellular immune functions of cervical cancer patients in comparison with healthy controls. It has been observed that the freshly isolated peripheral blood mononuclear cells (PBMC) exhibit natural cytotoxicity (NC) against a number of targets including tumor cells, mainly delivered by NK cells, which are non-adoptive and MHC unrestricted. Upon stimulation with cytokines like IL-2, IL-7, IL-12, IL-15 and interferons, PBMC acquire lymphokine activated killer (LAK) activity which enables them to lyse a wide range of targets including fresh tumor cells and virally infected cells. We compared the effect of IL-2 and IL-12 on enhancement of NC of PBMC from cervical cancer patients. IL-12 stimulated cultures (CD3+, CD56+) exhibited significant levels of tumoricidal activity. IL-2 stimulated lytic activity sustained even after 10 days while that of IL-12 stimulated cells declined after 6 days. Activation of PBMC was marked by increase in the expression of activation marker CD45RO and adhesion molecules LFA-1alpha, ICAM-1 and CD44. Addition of IL-12 to IL-2 stimulated cultures further enhanced the degree of lytic activity. Our data, thus, provide an evidence that PBMC from cervical cancer patients can be stimulated in response to cytokines and local or systemic treatment with low doses of cytokines may help to yield a better immune response against virus infected tumor cells in cervical cancer.     

Adhesion molecules on murine lymphokine-activated killer cells responsible for target cell killing: a role of CD2.

Lymphokine-activated killer (LAK) cells were induced from C57BL/6 mouse spleen cells and the effects of culture time on the expression of cell surface phenotypes and cytotoxic activity of LAK cells were determined. The expression of CD2 remarkably decreased after culture of LAK cells for 30 days, while LFA-1, a principal adhesion molecule in LAK cells, and CD3 were not changed by the culture. LAK cells cultured for 90 days completely lost CD2. In accordance with the decrease of CD2, the cytotoxic activity of LAK cells declined but a certain leven was retained even after the complete loss of CD2. The established LAK cell clones were also strongly positive for the expression of LFA-1 but negative for CD2. When the LAK cell clones were transfected with the CD2 cDNA, they started to express CD2 on their cell surface and to show greater binding ability and stronger cytotoxicity to target tumor cells. These results indicated that CD2 plays a role as an adhesion molecule responsible for target cell killing in murine LAK cells.