体外扩增CIK细胞杀伤胃癌细胞SGC-7901

目的：观察CIK细胞增殖情况，了解扩增后的最佳应用时机，并观察体外对SGC-7901的杀伤作用。方法：健康人外周血单核细胞（PBMC）在体外条件下经过多种细胞因子的共同刺激诱导成CIK细胞，计数培养不同时间的CIK细胞，用流式细胞术检测CIK细胞的表型特征。用MTT法检测杀伤活性，对比CIK细胞与5-FU对SGC-7901的杀伤作用，并观察了两者联合应用的效果。结果：CIK细胞随体外培养时间的延长，数量及杀伤活性均增加。体外培养20d增殖124倍，CD3^＋、CD56^＋双阳性细胞的比例达66．1％，其后两者数量增长缓慢；体外实验显示CIK细胞对胃癌SGC-7901细胞株有明显的杀伤作用，最高杀伤效率为73．13％，其杀伤作用优于5-FU，P〈0．05。二者联合应用杀伤作用降低。结论：CIK细胞具有较强的抗胃癌细胞活性；体外培养15～20d时应用较为合适；联合应用时5-FU可降低CIK细胞的杀伤效率。     

CD 40激发肿瘤抗原特异性DCs对CIK细胞生物学活性的影响

目的:研究CD40激发凋亡肿瘤细胞致敏的树突状细胞(dendritic cells,DCs)对细胞因子诱导杀伤(cytokine-inducedkiller,CIK)细胞的细胞表型、增殖活性及细胞毒活性的影响。方法:常规方法从健康人外周血单个核细胞中诱导DCs和CIK细胞,采用凋亡肿瘤细胞负载DCs,并用或不用激发型CD40单克隆抗体(CD40mAb)激活DCs成熟;成熟DCs与同源的CIK细胞共育5d,分别获得DC40Ag-CIK细胞及DCAg-CIK细胞;观察细胞增殖活性;流式细胞仪检测细胞表型;酶联免疫吸附实验(enzyme-linked immunosorbent assays,ELISA)法检测细胞培养上清液中IFN-γ的含量;[3H]-TdR掺入法测定细胞杀伤活性。结果:凋亡肿瘤细胞负载激发可使DCs上调表达CD1a、CD80、CD86、CD83、HLR-DR,联合CD40mAb激发可进一步促进DCs成熟;培养至第14天时,DC40Ag-CIK细胞、DCAg-CIK细胞、CIK细胞分别扩增(18.2±1.7)倍、(15.0±1.2)倍、(9.3±1.8)倍;DC40Ag-CIK细胞中的CD3 CD56 比例较DCAg-CIK细胞和CIK细胞明显上调(P<0.05);DC40Ag-CIK细胞、DCAg-CIK细胞对A549杀伤活性强于CIK细胞(P<0.05),并且DC40Ag-CIK细胞强于DCAg-CIK细胞(P<0.05);CIK细胞、DCAg-CIK细胞、DC40Ag-CIK细胞培养上清液中IFN-γ的含量递增,分别为(1494.7±246.3)pg/mL、(2706.3±197.0)pg/mL、(3676.3±335.0)pg/mL。结论:与单独凋亡肿瘤细胞负载的DCs相比,CD40激发的凋亡肿瘤细胞负载的DCs可进一步提高CIK细胞的增殖活性及细胞毒活性。     

抗CD3单抗和rIL-2共刺激诱生扩增人CD3AK的实验研究Ⅲ CD3AK与常规LAK细胞的比较

抗CD3单抗激活的杀伤细胞(Anti-CD3 monoclonal antibody activated killer cells．CD3AK)是用抗CD3单抗(Anti-monoclonal antibody，Anti-CD3 mAb)激活的一类杀伤细胞，具有较强的增殖能力和细胞毒活性。在CD3AK诱生扩增过程中除抗CD3单抗外也需要rIL-2作为生长因子，这就提出一个问题。     

荷肿瘤抗原的DC细胞增强CD3AK细胞特异杀伤作用的研究

目的 探讨肺癌细胞株A549的肿瘤冻融抗原（Ag）负荷脐血来源树突状细胞（DC）（Ag＋DC）与CD3AK细胞混合培养制备的Ag＋DC＋CD3AK对肺癌细胞株A549特异杀伤活性的影响。方法 用脐带血单个核细胞（UBMC）分别制备DC细胞、CD3AK细胞，用肺癌细胞株A549的制备肿瘤抗原（Ag），用Ag负荷DC制备Ag＋DC，再把Ag＋DC和CD3AK共培养制备Ag＋DC＋cD3AK细胞；用MTT法检测不同效应细胞：UBMC、CD3AK细胞，Ag＋CD3AK和Ag＋DC＋CD3AK细胞对A549肺癌细胞株的杀伤活性。结果 各种效应细胞对A549细胞的杀伤活性分别是：Ag＋DC＋CD3AK（62．11％）〉Ag＋CD3AK（50．34％）〉CD3AK（45．91％）〉UBMC（34．35％），而且Ag＋CD3AK组的杀伤活性高于CD3AK组（P〈0．05）；Ag＋DC＋CD3AK组的杀伤活性更明显的高于CD3AK组（P〈0．01）。结论 Ag能增强CD3AK细胞对亲本靶细胞的特异杀伤活性，肿瘤抗原负荷的DC（Ag＋DC）能进一步增强CD3AK细胞对亲本靶细胞的特异杀伤活性，为CD3AK细胞的特异免疫治疗的临床应用提供了基础资料。     

CD3和CD28单克隆抗体联合作用对CIK细胞及其亚群增殖及功能的影响

目的:探讨CD3和CD28单克隆抗体联合作用对细胞因子诱导的杀伤(cytokine-indueed killer,CIK)细胞及其亚群增殖及功能的影响.方法:采集2014年6月至2015年6月在郑州大学第一附属医院接受生物细胞治疗的20例肿瘤患者(肾癌6例,肺癌5例,肝癌、乳腺癌和恶性黑素瘤各2例,胆囊癌、淋巴瘤和卵巢癌各1例)的外周血,每例外周血均分为4组(对照组,单用CD3单抗组,单用CD28单抗组,CD3和CD28单抗联合作用组)进行CIK细胞的诱导培养.用CFSE染色检测CIK细胞的增殖能力;ELISA检测CIK细胞分泌细胞因子IFN-γ、TNF-α和IL-2的水平;磁珠分选CIK细胞亚群CD4+、CD8+和CD56+细胞,然后用ELISA检测对CIK细胞亚群分泌IFN-γ、TNF-α和IL-2能力的影响.结果:CD3/CD28单抗联合作用(PI =59.35)对CIK细胞的增殖能力的影响与单独使用CD3单抗(PI =64.4)相比效果并不明显,但是可以刺激CIK细胞分泌较高水平的IFN-γ和TNF-α[IFN-γ:(384.6 ±263.1)vs(201.5 ±271.5) pg/ml,P=0.0361;TNF-α:(4.795±1.251) vs(2.835±0.443) pg/ml,P=0.0265];进一步分析发现,两单抗的联合作用可增强亚群细胞CD8+分泌IFN-γ、CD56+分泌IL-2和TNF-α的活性,从而起到对淋巴细胞功能的增强作用.结论:CD3与CD28单抗联合作用不能增强CIK的增殖能力,但可以在一定程度上增强CIK细胞的功能,在肿瘤的细胞免疫治疗应用中具有一定的潜力.     

CIK细胞对慢性粒细胞白血病G0期CD34+白血病细胞的杀伤作用

目的研究细胞因子诱导杀伤细胞(cytokine induced killer,CIK)对慢性粒细胞白血病(chronic myeloid leukemia,CML)中G0期原始干细胞(CD34+)的杀伤作用.方法5例CML慢性期患者外周血标本在体外诱导培养CIK细胞,流式细胞仪(FCM)检测其表型.G带法检测CIK细胞核型.应用免疫磁珠技术分选CD34+CML白血病细胞,AO染色后利用FCM检测CIK细胞对G0期CD34+CML白血病细胞的杀伤作用.半固体培养法检测CIK细胞对正常骨髓CFU-GM、BFU-E集落的影响.结果CIK细胞在培养第3周时,CD3+细胞数量达(97.29±3.55)%,CD3+CD56+细胞绝对数量增加了356倍,染色体分析证实,CIK细胞为正常核型,流式细胞仪检测CIK细胞对G0期CD34+CML白血病细胞平均杀伤率为66.73%.对照组与加入CIK细胞组的CFU-GM集落数分别为150±16和145±20,差异无统计学意义,P=0.38;两组的BFU-E集落数分别为68±5和65±8,差异无统计学意义,P=0.23.集落类型、大小两组相似,差异无统计学意义,P=0.46.结论CIK细胞可从CML患者外周血中大量扩增,来源于正常淋巴细胞,对G0期CD34+CML白血病细胞有明显抑制作用,对正常造血前体细胞无抑制作用,可作为过继细胞免疫治疗的效应细胞治疗CML.     

可溶性结肠癌抗原与CD3mAb,IL-2联合诱导杀伤细胞的实验研究

IL-2诱导体外杀伤细胞扩增慢,高依赖IL-2,特异性差,影响了其疗效的发挥.根据报道CD3mAb与IL-2在杀伤细胞的诱导过程中有增强作用.为了获得具有高效,特异细胞毒活性的抗肿瘤效应细胞,我们采用CD3mAb、IL-2与可溶性肿瘤抗原(结肠癌抗原)联合诱导正常人外周血单个核细胞获得相对特异的杀伤细胞(TAK),并对TAK与LAK在增殖活性、杀伤活性及表型上进行了对比.     

来源于未缓解白血病患者的DC-CIK细胞对白血病细胞的杀伤活性

目的：体外诱导培养缓解期及未缓解期急性白血病患者来源的树突状细胞（dendritic cell, DC)联合细胞因子诱导的杀伤(cytokine-induced killer, CIK)细胞,比较两者的增殖能力、免疫表型及对白血病细胞的杀伤活性。方法: 提取2013年4月25日至2014年1月17日河北医科大学第二医院血液科收治的复发难治未缓解期白血病患者（7例）和缓解期患者（7例）的外周血单个核细胞,分别按常规方法诱导产生DC-CIK细胞,在培养过程中检测细胞增殖倍数,流式细胞术检测CD3+CD8+、CD3+CD56+细胞比例,细胞涂片法和流式细胞术检测培养前后CD34+白血病细胞比例,CCK-8法检测两组DC-CIK细胞对K562细胞及患者单个核细胞的杀伤活性。结果: 缓解组和未缓解组来源的DC-CIK细胞均以相似的速度快速增殖,CD3+CD8+和CD3+CD56+细胞比例均随培养时间延长显著升高（ P <0.05）,但两组间差异没有统计学意义（ P >0.05）。培养第15天时,非缓解组DC-CIK细胞涂片检测未见CD34+原始白血病细胞,流式细胞术检测显示CD34+细胞比例较培养前显著降低\[（0.1±0.05）% vs (8.3±3.1)%, P <0.05\]。效靶比在5 ∶1~20 ∶1范围内时,缓解组与未缓解组DC-CIK细胞对K562细胞的杀伤率差异没有统计学意义（ P >0.05）,但未缓解组DC-CIK对患者单个核细胞的杀伤率显著高于缓解组（ P <0.05）,并且显著高于未缓解组对K562细胞的杀伤率（ P <0.05）。结论:未缓解期白血病患者来源的DC-CIK细胞在增殖活性、CD3+CD8+和CD3+CD56+表达水平和对K562细胞的非特异性杀伤活性方面与缓解期患者来源的DC-CIK细胞均相似,但对患者单个核细胞的杀伤具有更强的特异性。     

恶性实体瘤患者自体细胞因子诱导杀伤细胞过继免疫治疗的安全性改进

目的观察恶性实体瘤患者外周血来源的细胞因子诱导杀伤细胞在自体血浆中的增殖特性及静脉回输治疗的安全性。方法用淋巴细胞分离液分离获取50例恶性实体瘤患者外周血单个核细胞,在含10%自体血浆的RPMI-1640培养液中添加rhIFN-γ、Anti-CD3mAb和rhIL-2联合诱导单个核细胞为细胞因子诱导杀伤细胞。在培养第13天进行细胞计数,通过流式细胞术检测细胞免疫表型,并将细胞进行自体回输。结果在培养第13天,细胞因子诱导杀伤细胞得到大量扩增,细胞数增加30.0±5.2倍。CD3 、CD3 CD4 、CD3 CD8 和CD3 CD16 CD56 细胞在培养第13天分别占(93.0±6.1)%、(28.4±7.2)%、(65.5±4.5)%和(25.8±12.2)%。恶性实体瘤患者回输自体细胞因子诱导杀伤细胞的治疗过程中未观察到明显的毒副作用,主要表现有畏寒、发热。结论恶性实体瘤患者血浆可以高效扩增自体细胞因子诱导杀伤细胞,临床应用安全可靠,且无明显的毒副作用。     

荷CEA-rV的DC增强CD3AK对CEA阳性肿瘤特异杀伤作用的研究

目的 观察荷CEA-rV的DC增强CD3AK细胞对CEA阳性肿瘤细胞的特异性杀伤功能。方法 用脐血制备脐血单个核细胞（UBMC），培养3h后，收集悬浮细胞制备CD3AK，重悬贴壁细胞制备DC。DC培养3d时加入CEA-rV，制备CEA-vV＋DC；在CD3AK培养8d时，加入CEA-rV＋DC混合培养，制备CEA-rV＋DC＋CD3AK。用MTT法测效应细胞UBMC，CD3AK，DC＋CD3AK，CEA-rV＋DC＋CD3AK，分别对CEA阳性靶细胞：LOVO，A549和CEA阴性靶细胞：肝癌细胞株BEL-7402，红白血病细胞株K652的杀伤活性。结果 ①用CEA兔抗人单克隆抗体对四种靶细胞株的鉴定结果表明，LOVO和A549确是CEA阳性细胞株，BEL．7402和K562确是CEA阴性细胞株。②培养10d的DC流式细胞仪检测结果示：高表达MHCI，Ⅱ类分子CD86（82．7％），CD80（51．1％），CD83（57．5％），CD40（69.4％），低表达CD123分子，光学和电子显微镜观察，DC细胞呈不规则形，有大量突起和伪足，成熟DC直径15-20um，胞浆线粒体，内质网丰富。证明为成熟DC。③CD3AK细胞和单纯UBMC细胞的流式细胞仪的免疫分子表型结果是，无论CD3，CD4，CD8和CD28，在CD3AK细胞组中的比率都是UBMC组的二倍以上。说明CD3AK组中成熟T淋巴细胞量明显高于UBMC细胞组。④三种效应细胞CEA-rV＋DC＋CD3AK，DC＋CD3AK和CD3AK对四种靶细胞的杀伤率均比单纯UBMC组明显提高（P〈0.01），而且CEA-rV＋DC＋CD，AK组〉DC＋CD3AK组〉CD3AK组〉UBMC组。说明细胞因子，DC和CEA-rV都有进一步提高UBMC广谱的杀伤肿瘤细胞活性的作用。⑤CEA-rV＋DC＋CD3AK和CD，AK相比较，对CEA阳性细胞LOVO和A549的杀伤活性提高的显著性（P〈0．01）明显优于对CEA阴性细胞K562和BEL-7402显著性（P〈0．05）。说明CEA-rV＋DC能显著提高CD3AK细胞对CEA阳性肿瘤的特异杀伤活性。CEA-rV＋DC＋CD3AK组和DC＋CD3AK组相比，对CEA阴性细胞株的杀伤活性无显著差异（P〉0．05），而对CEA阳性细胞株的杀伤活性确能明显提高（P〈0．01）。进一步证实了CEA-rV能明显提高CD3AK对CEA阳性肿瘤的特异杀伤作用。结论 CEA-rV＋DC能明显提高CD3AK细胞对CEA阳性细胞株杀伤作用，单纯DC对提高CD3AK杀伤CEA阳性细胞的作用不显著，结果表明CEA-rV在提高CD3AK对CEA阳性肿瘤的特异杀伤作用中起着关键作用。     

Bispecific Antibody-directed Antitumor Activity of Human CD4+ Helper/Killer T Cells Induced by Anti–CD3 Monoclonal Antibody plus Interleukin 2

Freshly isolated human CD4⁺ T cells can not respond to recombinant interlenkin 2 (rIL–2) because of their lack of p75 IL–2 receptor expression. However, we succeeded In inducing a marked proliferation of purified CD4⁺ T cells by activation with rIL–2 plus anti–CD3 monoclonal antibody (mAb) cross–linked to a plastic plate. The proliferated CD4⁺ T cells produced a significant amount of IL–2 upon stimulation with phorbol ester plus A23187. Interestingly, CD4⁺ T cells activated with anti–CD3 mAb plus rIL–2 revealed a strong cytotoxic activity against Fc receptor (FcR)–positive tumor cells in the presence of anti–CD3 mAb. Moreover, the CD4⁺ T cells could lyse FcR–negative glioma cells by targeting with bispecific mAb containing anti–CD3 mAb and anti–glioma mAb. Thus, we demonstrated that rIL–2 and immobilized anti–CD3 mAb allowed the rapid generation of human CD4⁺ helper/killer T cells, which may be useful for the development of a new adoptive tumor immunotherapy.     

Phenotype and function of a CD56+ peripheral blood monocyte.

G-CSF primed CD34 cells cultured for 2-3 weeks in IL-2 and stem cell factor generate CD56(high) cells with phenotypic and morphologic features of NK cells, and a novel adherent CD56(low) CD16- population expressing myeloid markers (CD33 and HLA-DR). We hypothesized that similar cells might also occur in peripheral blood. In 13/13 normal individuals, we found a circulating population of CD56(low), CD33+, FcgammaRI+, FcgammaRII+, HLA-DR+, CD11b(high), CD14+ monocytes closely resembling the cultured CD56(low)CD33+ cells. They may represent a normal counterpart of the CD56+ CD33+ hybrid myeloid/natural killer cell leukemia. Their mean frequency was 1.3+/-1% (standard deviation), range 0.16-3.5%, of total mononuclear cells. CD56(low)CD33+ cells, primed with cytomegalovirus antigen, induced autologous T-lymphocyte proliferation comparably to CD56-, CD14+ peripheral blood monocytes (PBM). Conversely, CD56(low) cells induced greater T-cell proliferation than CD56- PBM when lymphocyte responders were HLA mismatched. Unstimulated CD56(low)CD33+ cells showed a low antiproliferative effect on K562, which was increased upon LPS stimulation. The pattern of cytokine production by CD56(low)CD33+ cells and PBM largely overlapped; however, they produced detectable levels of IL-6 and IL-1beta. These results define a minor monocyte population with distinct phenotypic and functional features.     

Bispecific antibody-directed antitumor activity of human CD4+ helper/killer T cells induced by anti-CD3 monoclonal antibody plus interleukin 2.

Freshly isolated human CD4+ T cells can not respond to recombinant interleukin 2 (rIL-2) because of their lack of p75 IL-2 receptor expression. However, we succeeded in inducing a marked proliferation of purified CD4+ T cells by activation with rIL-2 plus anti-CD3 monoclonal antibody (mAb) cross-linked to a plastic plate. The proliferated CD4+ T cells produced a significant amount of IL-2 upon stimulation with phorbol ester plus A23187. Interestingly, CD4+ T cells activated with anti-CD3 mAb plus rIL-2 revealed a strong cytotoxic activity against Fc receptor (FcR)-positive tumor cells in the presence of anti-CD3 mAb. Moreover, the CD4+ T cells could lyse FcR-negative glioma cells by targeting with bispecific mAb containing anti-CD3 mAb and anti-glioma mAb. Thus, we demonstrated that rIL-2 and immobilized anti-CD3 mAb allowed the rapid generation of human CD4+ helper/killer T cells, which may be useful for the development of a new adoptive tumor immunotherapy.     

Transient depletion of CD4+ T cells augments IL‐21‐based immunotherapy of disseminated neuroblastoma in syngeneic mice

IL‐21 is a member of the IL‐2 cytokine family, produced by CD4+ T cells. We previously showed that immunotherapy (IT) with IL‐21‐transduced neuroblastoma cells (Neuro2a/IL‐21) cured 33% of syngeneic mice bearing systemic NB. Here, we studied whether the removal of Treg cells could potentiate the therapeutic efficacy of Neuro2a/IL‐21 vaccine. The administration of anti‐CD25 mAb, which targets Treg cells, slightly potentiated the effect of vaccine IT (50% cure rate), but anti‐CD4 mAb had a more potent effect leading to 80% cure rate. Anti‐CD25 mAb, indeed, only partially depleted CD4+CD25+FoxP3+ Treg cells, whereas anti‐CD4 mAb was more effective in this respect, leading to 90% depletion of Treg cells. In mice receiving vaccine+anti‐CD4 mAb, which developed systemic immunity to NB, CD4+ T cells counts completely recovered in 90 days. Depletion of CD8+ T cells abrogated the effect of the combined IT, indicating a predominant role of these cells in driving the immune response. In addition, CD8+ T cells from cured mice coinjected with Neuro2a/parental cells (pc) in NOD‐SCID mice completely inhibited tumor growth. Spleen cells from mice receiving Neuro2a/IL‐21 vaccination showed increased expression of IFN‐alpha2, ‐beta1 and ‐gamma mRNA. Moreover, mice receiving vaccine therapy alone or vaccine+anti‐CD4 mAb showed increased IFN‐gamma serum levels and IFN‐gamma‐producing CD8+ T cells were found in spleen cells. In conclusion, anti‐CD4 mAb potentiated IL‐21‐based IT by removing Treg cells and/or their precursors and other potentially immune‐suppressive CD4+ cell subsets, thus allowing the development of an IL‐21‐driven CD8+ T cell response, which mediates NB rejection.     

Sequential Involvement of Two Distinct CD4+ Regulatory T Cells during the Course of Transplantable Tumor Growth and Protection from 3–Methylcholanthrene-induced Tumorigenesis by CD25–depletion

The involvement of two phenotypically different regulatory T cells in different stages of tumor growth was investigated. Treatment of BALB/c mice with anti-CD25 monoclonal antibody (mAb) (PC61), but not anti-CD4 mAb (GK1.5) before RL male 1 or Meth A inoculation caused tumor rejection. On the other hand, treatment of BALB/c mice with anti-CD4 mAb (GK1.5) but not anti-CD25 mAb (PC61) on day 6 after inoculation of the same tumors caused rejection. The findings suggest that CD4⁺CD25⁺ T cells downregulated the rejection response in the early stage of tumor growth. On the other hand, putative CD4⁺CD25⁻ T cells downregulated the tumor rejection response in the late stage. Both CD4⁺CD25⁺ and putative CD4⁺CD25-T cells appeared to inhibit the efficient generation of cytotoxic T lymphocytes (CTL). The present study also demonstrated that the treatment of BALB/c mice with anti-CD25 mAb (PC61) at 4 or 6 weeks after 3–methylcholanthrene (3–MC) inoculation retarded tumor occurrence and prolonged survival.     

重组人纤维连接蛋白对肝细胞肝癌患者自体CIK细胞体外扩增及生物学活性的影响

目的观察重组人纤维连接蛋白(RN)对肝细胞肝癌(HCC)患者细胞因子诱导的杀伤细胞(CIK)生物活性的影响,建立一种高效的CIK细胞体外扩增方法。方法取15份HCC患者外周血,分离单个核细胞,分别使用传统方法(抗CD3 mAb+IFN-γ+IL-2)和RN法(RN+抗CD3 mAb+IL-2)诱导,观察不同时间点两组细胞形态、数目、细胞表型和杀伤效率。结果与传统方法相比,RN组显著增加CIK细胞扩增倍数(P<0.05),是对照组的2.0~2.8倍,提高活化T细胞(CD25+T细胞)数量,增加CD3⁺CD8⁺T细胞比例,增强对肝癌细胞株的杀瘤活性。结论RN能够高效促进HCC患者CIK细胞的扩增,可以替代传统方法。     

淋巴结来源的CD3AK细胞的增殖和表型变化分析

目的：研究肺癌清扫淋巴结的淋巴细胞，经CD3单克隆抗体激活后的杀伤细胞（CD3AK细胞）在体外的增殖和表型变化。方法：取肺癌清扫淋巴结，用机械方法分离成单细胞悬液，加CD3单克隆抗体和小剂量IL2共同培养，每隔1天用流式细胞术进行细胞表型分析及用台盼蓝活细胞计数法观察细胞增殖情况。结果：表型分析显示CD3，CD8，CD56，CD25增加。培养第8天淋巴细胞增殖达高峰，数量是培养前的2.33倍。结论：CD3单克隆抗体能明显激活各种T细胞增殖，使CD3AK细胞维持杀伤功能。     

DC与CIK共培养对骨肉瘤细胞的杀伤作用

目的探讨树突状细胞(dendritic cells,DC)与细胞因子诱导的杀伤细胞(cytokine-induced killer cells,CIK)共培养对人骨肉瘤细胞的杀伤效应。方法从健康志愿者中分离外周血单个核细胞,制备DC和CIK,并以5∶1比例混合培养。采用CCK-8法和流式细胞术检测DC-CIK共培养对人骨肉瘤HOS细胞增殖和凋亡的影响;构建裸鼠移植瘤模型,观察DC-CIK共培养对裸鼠体内移植瘤生长的影响。结果流式细胞术检测结果显示,分化成熟的DC中CD83+和CD86+阳性细胞比例均高于未成熟的DC(P=0.009,0.006);DC-CIK共培养后,CD3+CD8+和CD3+CD56+阳性细胞比例均高于CIK单独培养组(P=0.004,0.004)。与DC-CIK共培养后,HOS细胞增殖能力受抑制,细胞凋亡能力增强(P<0.05);PD-L1的表达水平明显下调(P=0.006)。裸鼠移植瘤模型中,DC-CIK共培养可抑制HOS细胞诱导的移植瘤生长(P=0.008)。结论DC-CIK共培养能抑制人骨肉瘤HOS细胞增殖并诱导细胞凋亡,其作用机制可能与靶向调控PD-1/PD-L1信号通路有关。     

The Roles of CD8+ and CD4+ Cells in Tumor Rejection

In vivo administrations of anti-Lyt-2.2 (CDS) mAb and anti-L3T4 (CD4) mAb selectively eliminated CD8⁺ cells amd CD4⁺ cells, respectively. The relative potencies of CD8⁺ cells and CD4⁺ cells and their roles in primary tumor rejections were studied by investigating the effects of these mAbs on tumor growth. CD8⁺ cells were themselves fully capable of mediating rejection in 5 different tumor rejection systems: two radiation leukemia virus (RadLV)-induced leukemias, B6RV2 and BALBRVD, a radiation-induced leukemia BALBRL♂1, and a plasmacytoma BALBMOPC-70A in CB6F₁ mice, and a Friend virus-induced leukemia B6FBL-3 in B6 mice. On the other hand, CD4⁺ cells were capable of resisting tumor growth of B6FBL-3, but not of the other four tumors. Furthermore, for efficient rejection of CB6F₁UV+^˚l sarcoma by CB6F₁ mice, synergy of CDS⁺ and CD4⁺ cells was necessary. Blocking of UV+^˚ 1 rejection was abrogated by delayed administration of anti-L3T4 (CD4) mAb but not anti-Lyt-2.2 (CDS) mAb, indicating the involvement of CD4⁺ cells in only the initial phase of rejection.