FL联合TPO体外培养AC133+细胞表面标记的变化

目的:探讨脐血造血干/祖细胞的体外扩增.方法:免疫磁珠法分离纯化脐血AC133+细胞,在细胞因子FLT3配体、血小板生成素作用下,体外扩增,检测有核细胞扩增的倍数.采用流式细胞仪分析细胞表面标志的变化.结果:FLT3联合血小板生成素体外培养2周.有核细胞扩增(18±8)倍.CD34细胞扩增2.8倍,AC133+未获扩增,CD34细胞纯度由(56±23)%下降到(8±1)%,AC133+细胞由85%下降到(0.1±0.1)%.随着体外培养时间延长至4周,有核细胞,CD34+进一步扩增.分别扩增475倍和17倍.但细胞随之发生分化,CD34+细胞占有核细胞中的比例下降至2%,AC133+细胞消失.CD45细胞上升到1.3%.结论:FL联合TPO长期培养AC133+细胞能使CD34细胞扩增.     

脐血造血干/祖细胞体外扩增实验研究

目的:探讨人脐血造血干/祖细胞(hematopoietic stem/progenitor cells,HSC/HPC)在适合临床移植条件下体外扩增以获得理想的造血干/祖细胞数量的最佳细胞因子组合及扩增时间,为脐血经体外扩增后成为高体重受者克服脐血移植细胞数量不足及缩短植入时间提供客观指标。方法:采集脐血4份,分离单个核细胞(mononuclear,MNC),免疫磁珠分选(MiniMACS)CD+34细胞,分别在细胞因子组合为A:SCF+FL+TPO、B:SCF+FL+TPO+IL蛳3及C:SCF+FL+TPO+G蛳CSF支持的无血清、无基质的液体培养基中扩增培养14 d,收集扩增前、扩增后7 d及14 d的细胞进行细胞表型分析、半固体集落培养及端粒酶活性分析。结果:在含细胞因子IL蛳3组扩增培养7 d及14 d,均获得了总有核细胞数(total nucleated cells,TNC)及集落形成细胞(colony蛳forming cell,CFC)最大的扩增倍数。扩增7 d,IL蛳3组的CD+34细胞扩增倍数为(10.11±7.21 )倍,较其他两组稍低,但无统计学意义;三组CD+33细胞、CD+41细胞、CD+71细胞也得到了较大程度的扩增,IL蛳3组高于其他两组;3组扩增后的细胞端粒酶活性均上调,7 d时A值达最高,IL蛳3组低于G蛳CSF组(P =0.008)。随着扩增时间的延长,扩增后的细胞端粒酶活性下降;CD+34细胞占TNC的比例也明显下降,部分定向祖细胞的比例也出现先升高后     

rhIL-17对小鼠造血前体细胞和人脐血CD34+干细胞分化发育的影响 *

目的:探讨重组人白细胞介素-17(Interleukin-17,IL-17)对小鼠骨髓造血前体细胞和人脐血来源的CD34~ 干细胞生长发育的影响.方法:采用常规方法采集小鼠造血前体细胞;采用Mini-MACS分离技术,从正常人脐血分离人CD34~ 干细胞.体外加入IL-17和/或GM-CSF、IL-4培养分离的前体细胞,应用流式细胞仪检测其表型,采用ELISA法检测了其分泌的IL-12水平,通过[~3H]-TdR掺入法测定其刺激同种异体T淋巴细胞增殖的能力.结果:IL-17促进了小鼠骨髓来源的未成熟DC表达Ia,B7-2等免疫分子,促使其分泌较高水平的IL-12,该细胞也能刺激同种异体T细胞有效增殖,表现出了成熟DC的特征.IL-17单独培养9d促使人脐血CD34~ 干细胞扩增了2倍,部分细胞高表达CD1a及B7-2,低表达HLA-DR,未检测到CD83的表达.该细胞能促使同种异体T细胞增殖,但作用较弱;而rhIL-17与GM-CSF联合培养后扩增了14倍,培养细胞中CD1a、B7-2阳性细胞的比例明显升高,且此细胞刺激同种异体T细胞增殖的能力较强.结论:IL-17体外可促进小鼠骨髓造血前体细胞来源的DC成熟;与GM-CSF联合培养既能促进CD34~ 干细胞增殖,又能使之获得DC特征,初步提示IL-17与GM-CSF联合作用可促进CD34~ 干细胞向DC分化.     

IL-6/sIL-6R对人脐血CD34+细胞体外扩增的作用

背景和目的:造血干细胞具有自我更新、多向分化与重建长期造血的潜能,因此,造血干细胞广泛应用于干细胞移植、免疫治疗、基因治疗等领域.胎儿脐带血中富含造血干细胞,但单份脐带血中造血干细胞的数量有限,不能充分满足临床和科研需要,因此在体外培养使脐带血干/祖细胞数量扩增的研究日益受到重视.已知一些细胞因子可以在体外培养中使脐血干/祖细胞大量扩增.新近发现IL-6/sIL-6R(可溶性IL-6受体)或其融合蛋白可促使脐带血CD34+细胞中CD34+gp130+IL-6R-细胞亚群在体外培养中大量扩增.本实验旨在观察IL-6/sIL-6R在脐带血CD34+细胞体外扩增中的作用,并探讨合适细胞因子组合.方法:脐血CD34+细胞用Mini MACS分离,然后在含有不同细胞因子组合的液体培养基中体外培养7天或14天,培养前后分别进行有核细胞计数、用FCM(流式细胞术)测定CD34+细胞比例计算CD34+细胞总数及进行CFU-GM集落培养.根据不同细胞因子组合分为空白对照组,SCF组,SCF+IL-6/sIL-6R组,SCF+FL+IL-6/sIL-6R组,SCF+FL组.结果:空白对照组和SCF组CD34+细胞数量在培养7天或14天后明显下降.SCF+IL-6/sIL-6R组培养7、14天分别使有核细胞及CD34+细胞绝对数扩增(7.1±2.4)倍、(39.0±14.0)倍及(1.8±0.7)倍、(4.8±2.4)倍;SCF+FL+IL-6/sIL-6R组(16.5±5.7)倍、(110.0±28.0)倍及(3.5±1.5)倍、(10.2±4.2)倍;SCF+FL组(17.3±3.8)倍、(104.0±21.0)倍及(3.6±2.1)倍、(8.4±3.5)倍.上述三组与对照组及SCF组差异均有显著性(P＜0.01),其中SCF+FL+IL-6/sIL-6R组和SCF+FL组扩增效果优于SCF+IL-6/sIL-6R组(P＜0.01),但是SCF+FL+IL-6/sIL-6R组和SCF+FL组之间差异无显著性(P＞0.05).增加sIL-6R浓度,当sIL-6R浓度为400 ng/ml时,细胞培养7天,SCF+FL+IL-6/sIL-6R组分别使有核细胞及CD34+细胞绝对数扩增(24.0±4.8)倍、(5.6±1.2)倍,优于SCF+FL组(P＜0.05).结论:IL-6/sIL-6R可以和SCF、FL产生协同作用,使人脐血CD34+细胞在体外扩增、并维持其分化潜能.但这种协同作用对sIL-6R的浓度存在一定依赖性.     

IL-6/sIL-6R对人脐血CD34+细胞体外扩增的作用

背景和目的:造血干细胞具有自我更新、多向分化与重建长期造血的潜能,因此,造血干细胞广泛应用于干细胞移植、免疫治疗、基因治疗等领域.胎儿脐带血中富含造血干细胞,但单份脐带血中造血干细胞的数量有限,不能充分满足临床和科研需要,因此在体外培养使脐带血干/祖细胞数量扩增的研究日益受到重视.已知一些细胞因子可以在体外培养中使脐血干/祖细胞大量扩增.新近发现IL-6/sIL-6R(可溶性IL-6受体)或其融合蛋白可促使脐带血CD34+细胞中CD34+gp130+IL-6R-细胞亚群在体外培养中大量扩增.本实验旨在观察IL-6/sIL-6R在脐带血CD34+细胞体外扩增中的作用,并探讨合适细胞因子组合.方法:脐血CD34+细胞用Mini MACS分离,然后在含有不同细胞因子组合的液体培养基中体外培养7天或14天,培养前后分别进行有核细胞计数、用FCM(流式细胞术)测定CD34+细胞比例计算CD34+细胞总数及进行CFU-GM集落培养.根据不同细胞因子组合分为空白对照组,SCF组,SCF+IL-6/sIL-6R组,SCF+FL+IL-6/sIL-6R组,SCF+FL组.结果:空白对照组和SCF组CD34+细胞数量在培养7天或14天后明显下降.SCF+IL-6/sIL-6R组培养7、14天分别使有核细胞及CD34+细胞绝对数扩增(7.1±2.4)倍、(39.0±14.0)倍及(1.8±0.7)倍、(4.8±2.4)倍;SCF+FL+IL-6/sIL-6R组(16.5±5.7)倍、(110.0±28.0)倍及(3.5±1.5)倍、(10.2±4.2)倍;SCF+FL组(17.3±3.8)倍、(104.0±21.0)倍及(3.6±2.1)倍、(8.4±3.5)倍.上述三组与对照组及SCF组差异均有显著性(P＜0.01),其中SCF+FL+IL-6/sIL-6R组和SCF+FL组扩增效果优于SCF+IL-6/sIL-6R组(P＜0.01),但是SCF+FL+IL-6/sIL-6R组和SCF+FL组之间差异无显著性(P＞0.05).增加sIL-6R浓度,当sIL-6R浓度为400 ng/ml时,细胞培养7天,SCF+FL+IL-6/sIL-6R组分别使有核细胞及CD34+细胞绝对数扩增(24.0±4.8)倍、(5.6±1.2)倍,优于SCF+FL组(P＜0.05).结论:IL-6/sIL-6R可以和SCF、FL产生协同作用,使人脐血CD34+细胞在体外扩增、并维持其分化潜能.但这种协同作用对sIL-6R的浓度存在一定依赖性.     

CIK的体外增殖及体内外杀瘤活性的实验研究

目的:从人骨髓造血前体细胞体外培养扩增树突状细胞(dendritic cells,DCs),测定其表型及T细胞刺激活性.方法:采用Mini-MACS分离技术,从正常人骨髓、脐血分离CD34~ 造血干细胞,体外以重组hGM-CSF,hTNF-α,hIL-3诱导培养2周,流式细胞术检测扩增细胞的表面表型及细胞内IL-12的表达,体外同种混合淋巴细胞反应检测扩增DCs的T细胞刺激活性.结果:从正常人骨髓、脐血分离得到高纯度(>90%)的CD34~ 造血干细胞,经重组hGM-CSF,hTNF-α的共同诱导培养,扩增得到大量DCs,加人hIL-3可以进一步增加DCs产量;FACS检测表明,扩增的DCs表达HLA-DR,CD40,CD54,CD80,CD86分子,细胞内有hIL-12的P35,P40亚基的表达;与外周血单核细胞培养生成的DCs相比,由CD34~ 干细胞扩增的DCs具有更强的激发同种T细胞增殖的能力.结论:人CD34~ 干细胞体外经诱导培养,可以生成大量功能成熟的DCs,从而为进一步开展DCs的基础及临床研究打下了基础.     

人脐血树突状细胞抗神经胶质瘤的体外免疫效应研究

目的　研究肿瘤抗原致敏的树突状细胞 (DC)对神经胶质瘤细胞的免疫杀伤效应。方法　应用免疫磁珠分选脐血 CD34细胞 ,经 SCF FL3 GM- CSF IL- 4 TNF- α的联合诱导培养 DC,采用相差显微镜观察树突状细胞的形态 ;流式细胞仪作 DC的表面标志检测 ;MTT比色法测定同种异型的混合淋巴细胞反应能力和诱导CTL 毒性的检测。结果　 (1)脐血 CD34细胞在体外经细胞因子联合刺激后呈典型的 DC形态 ;(2 )流式检测 CD4 0、CD80和 CD86等成熟 DC特异性表面标志呈高表达 ,分别与诱导前比较 ,差异均有显著性 ;(3) MTT法测得脐血来源的 DC较外周血 DC刺激同种异体 T淋巴细胞增殖能力弱 ;经肿瘤抗原负载的 DC体外诱导出较强 CTL 毒性。结论　脐血 CD34细胞经体外扩增诱导的 DC具有典型的 CTL 毒性 ,为临床应用脐血来源 DC抗神经胶质瘤的生物治疗提供了理论依据     

脐带血CD+34细胞体外扩增优化条件的研究

 目的　分析比较不同细胞饲养层、不同刺激因子、脐血CD+34 细胞的纯度和含量等因素对脐血CD+34 细胞在体外扩增的影响,以筛选其体外扩增的优化方案。方法　采用正交实验,通过MTT法、细胞计数法及流式细胞分析技术测定脐血CD+34 细胞的增生情况;并采用以上方法及半固体培养体系对该细胞优化条件下的增生情况及集落形成能力进行检测。结果　优化条件下脐血CD+34细胞增生10倍,明显高于对照组的2.8倍（P＜0.01）,集落形成（CFU-C 36.67±6.11）也较对照组强（CFU-C 16.33±1.53）（P＜0.01）。结论　该培养系统能促进脐血CD+34 细胞体外扩增并能较好保持脐血CD+34 细胞的干细胞性质。     

脐血CD34^＋细胞体外扩增及树突状细胞诱导的实验研究

目的 研究脐血CD34^＋细胞在体外经造血细胞生长因子扩增后,诱导树突状细胞（DC）并观察该类DC在抗肿瘤免疫中的作用。方法 Ficoll分离脐血单个核细胞,免疫微磁珠法分离纯化CD34^ 细胞,以干细胞因子、flt3配体、白细胞介素－3和红细胞生成素体外扩增2周,诱导DC生成,观察肿瘤抗原负载后诱导特异性细胞毒T淋巴细胞生成和对肿瘤细胞杀伤作用。结果（1）脐血经免疫微磁珠法分离可获得高纯高的CD34^ 细胞;（2）2周体外扩增后,细胞数显著增加达150倍;体外集落试验证实该类细胞可形成粒单细胞集落形成单位／（CFU－GM）;（3）扩增的细胞可成功诱导成DC,并具有活化异体淋巴细胞的功能,负载肿瘤抗原后能诱导发生肿瘤特异性淋巴细胞,并可特异性杀伤Daudi肿瘤细胞。结论 脐血来源CD34^ 细胞在体外能成功地扩增,并诱导产生功能性DC。     

卵巢癌肿瘤浸润淋巴细胞某些细胞因子基因表达的研究

目的:研究卵巢癌肿瘤浸润淋巴细胞在体外扩增后某些细胞因子的表达.方法:利用RT-PCR和免疫学方法,分离5例刚分离的上皮性卵巢癌肿瘤浸润淋巴细胞(TIL)和其中3例腹水标本中肿瘤相关淋巴细胞(TAL),检测其在体外扩增后,IL-2,IL-2R,TNF-α,IFN-γ,IL-6等细胞因子基因的表达的水平与变化;测定细胞培养上清液中细胞因子的活性,分析TIL或TAL扩增前后杀伤肿瘤细胞的能力和免疫学特性的变化.结果:TIL在扩增前后的4周时间内,仅TNF-α mRNA基因表达是持续的,并可维持相当长一段时间;IFN-γmRNA基因的表达缺乏;IL-2mRNA基因表达只是一过性.体外加入抗CD3单抗或PHA(合适浓度)刺激TIL或TAL,IL-2,TNF-α,IhN-γmRNA表达明显增强.和TIL细胞因子基因表达不尽相同的是,TAL表达IL-6 mR-NA.结果还发现,体外IL-2的浓度对TIL的免疫学特性有很大的影响.结论:TIL在体外经rIL-2扩增后,免疫活性有明显提高.TIL的抗肿瘤活性在很大程度上依靠其分泌的多种细胞因子.     

Expression of caspase-3 in cord blood CD34<Superscript>+</Superscript> cells during culture in vitro

Objective: To investigate the expression and significance of caspase-3 protein in CD34⁺ cells from cord blood (CB) during culturein vitro with different growth factors.Methods: RT-PCR, Western blot and flow cytometry techniques were used to detect the expression of caspase-3 in CD34⁺ CB cells during culturein vitro. Results: Caspase-3 mRNA was constitutively expressed at a low level in freshly isolated CD34⁺ cells. The expression of caspase-3 mRNA and protein was upregulated when these cells were first expanded in suspension culture with growth factors for 3 days. However, only the 32 kDa inactive caspase-3 proenzyme was detected in the freshly isolated CD34⁺ cells as well as during the first 3 days expansion with cytokines. With longer culture timein vitro, especially in the presence of the combination of IL-3, IL-6 and GM-CSF, caspase-3 was activated and a cleavage product of 20 kDa became detectable.Conclusion: Caspase-3 is involved in apoptosis of primitive CB CD34⁺ cells during expansionin vitro. The study was supported by a grant from the National Natural Science Foundation of China (No. 39928010)     

Preferential expression of the vasoactive intestinal peptide (VIP) receptor VPAC1 in human cord blood-derived CD34+CD38- cells: possible role of VIP as a growth-promoting factor for hematopoietic stem/progenitor cells.

Primitive hematopoietic progenitor cells such as severe combined immunodeficiency- repopulating cells and long-term culture-initiating cells are enriched in CD34+CD38- cells derived from various stem cell sources. In this study, to elucidate the features of such primitive cells at the molecular level, we tried to isolate genes that were preferentially expressed in umbilical cord blood (CB)-derived CD34+CD38- cells by subtractive hybridization. The gene for VPAC1 receptor, a receptor for the neuropeptide vasoactive intestinal peptide (VIP), was thereby isolated and it was shown that this gene was expressed in both CD34+CD38- and CD34+CD38+ CB cells and that the expression levels were higher in CD34+CD38- CB cells. Next, we assessed the effects of VIP on the proliferation of CD34+ CB cells using in vitro culture systems. In serum-free single-cell suspension culture, VIP enhanced clonal growth of CD34+ CB cells in synergy with FLT3 ligand (FL), stem cell factor (SCF), and thrombopoietin (TPO). In serum-free clonogenic assays, VIP promoted myeloid (colony-forming unit-granulocyte/macrophage (CFU-GM)) and mixed (CFU-Mix) colony formations. Furthermore, in Dexter-type long-term cultures, VIP increased colony-forming cells at week 5 of culture. These results suggest that VIP functions as a growth-promoting factor of CB-derived hematopoetic progenitor cells.     

CD34high+ CD38(low/-) cells generated in a xenogenic coculture system are capable of both long-term hematopoiesis and multiple differentiation.

CD34+ cells isolated from human umbilical cord blood (HUCB) are thought to have potential in clinical applications such as transplantation and gene therapy. Recently, we developed a xenogenic coculture system involving HUCB-CD34+ cells and murine bone marrow stromal cells, HESS-5 cells, in combination with human interleukin-3 and stem cell factor. Under these xenogenic coculture conditions, the numbers of CD34high+ cells and primitive progenitor cells, such as CD34high+ CD38(low/-) cells and high proliferative potential colony-forming cells (HPP-CFCs), increased dramatically by a factor of 102.1, 66.5 and 104.9, respectively. In the present study, we used a secondary culture of B progenitor cells and long-term culture (LTC)-initiating cells to characterize and compare the progenitor capability of re-isolated CD34high+ CD38(low/-) cells, which have been identified as one of the most primitive progenitor cells, with that of freshly isolated CD34high+ CD38(low/-) cells. Compared with freshly isolated CD34high+ CD38(low/-) cells, the re-isolated CD34high+ CD38(low/-) cells were equally as capable of proliferating and differentiating into myeloid and B progenitor cells. No significant differences were observed in the frequency of LTC-initiating cells in the re-isolated CD34high+ CD38(low/-) cells compared with that in freshly isolated CD34high+ CD38(low/-) cells. Furthermore, the re-isolated CD34high+ CD38(low/-) cells were capable of long-term reconstitution and multiple differentiation in non-obese diabetic mice with severe combined immunodeficiency disease (NOD/SCID mice). The results demonstrate that this xenogenic coculture system can be used for successful in vitro expansion of HUCB-progenitor cells that possess the capability for both long-term hematopoiesis as well as multipotent differentiation into myeloid and lymphoid cells both in vivo and in vitro.     

脐血体外扩增干祖细胞和同时诱导分化T、NK和DCs免疫细胞

Umbilical cord blood stem cell transplantation (CB- SCT) has approached significant success in treatment of lethal congenital or malignant disorders, but CBSCT with low incidence of GVHD is associated with higher rates of delayed or failed engraftment and relapse than bone marrow transplants. This may be caused by immature im- mune cells, compared with the corresponding cells from adults. More studies have been reported that cord blood T lymphocytes are immature in phenotype and function…     

脐血CD34~+细胞扩增中系特异性分化抗原的变化

目的：探讨CB CD34^ 细胞扩增的最佳HGF组合及移植时机。方法：CB CD34^ 细胞培养于无血清培养液中,分别加入下列HGF,A组：对照组（无HGF）;B组：SCF,IL－3,IL－6;C组：SCF,IL－3,IL－6,G－CSF,Epo;D组：SCF,IL－3,IL－6,TPO,Flt3-L;E组：SCF,IL－3,IL－6,TPO,Flt3-L,G-CSF,Epo。培养22d,对不同培养时间的细胞进行NC数量及系特异性CD动态观察。结果：同对照组相比,扩增组NC和CD34^ 细胞均有不同程度的扩增。NC和CD34^ 细胞扩增高峰分别在第10天和第6天。E组的扩增效果最佳。CD检测显示,各组CD34^ 及CD34^ CD^38-细胞的比例逐渐减少,但B,D2组的CD34^ CD38^-细胞的比例在第6天有一过性回升,CD154^ ,CD13^ ,CD61^ 及Gly-A^ 细胞的比例则逐渐升高,D组CD34^ 及CD34^ ,CD38^-细胞的比例明显地高于E组（P＜0．01）,CD3^ 和CD19^ 细胞的比例在扩增中呈下降趋势,结论：HSPC分化程度与HGF组合及培养时间有关。就细胞总数而言,E组HGF组合最佳,但就HSPC含量来说,以D组为优,第1周扩增产物中HSPC含量较高,移植时间以扩增的第6－10天为宜。     

Cytoplasmic expression of EGFP in dendritic cells transfected with in vitro transcribed mRNA or cellular total RNA extracted from EGFP expressing leukemia cells

The present study was designed for identifying the protein synthesis in cytoplasm of dendritic cells transfected with in vitro transcribed mRNA and cellular total RNA extracted from tumor cells. Dendritic cells were generated from cord blood-CD34⁺ cells by culture with GM-CSF, SCF, and TNF-α, or from peripheral blood adherent cells or CD14⁺ cells by culture with GM-CSF and IL-4. Dendritic cells were transfected with in vitro transcribed EGFP mRNA or cellular total RNA, which was isolated from EGFP expressing K562, by electroporation using a square-wave pulse. Optimal in vitro transcribed EGFP mRNA transfection efficiency (>90%) was observed in a single electroporation of 1.75 kV/cm (electric field strength) with a pulse width of 250 μs. Although the intensity of EGFP expression in dendritic cells transfected with cellular total RNA was less compared with that in dendritic cells transfected with in vitro transcribed EGFP mRNA, a definite cytoplasmic synthesis of EGFP was demonstrated in dendritic cells transfected with cellular total RNA. The visual identification of cytoplasmic expression of cellular total RNA in dendritic cells revealed that electroporation of tumor cell-derived RNA could be a useful tool to load dendritic cells with tumor antigens for establishing an efficient dendritic cell-based tumor immunotherapy.     

The type of stromal feeder used in limiting dilution assays influences frequency and maintenance assessment of human long-term culture initiating cells.

The goal of this study was to evaluate if differences in culture conditions used in long-term culture assays affect enumeration of LTC-IC in freshly sorted or ex vivo expanded CD34+/HLA-DRdim/CD2-/CD7- (34+/Lin-) cells. The variables examined included different stromal feeders (murine bone marrow fibroblast cell line, M2-10B4 and murine fetal liver cell line, AFT024) and presence or absence of cytokines (MIP-1alpha + IL-3). The absolute LTC-IC frequency in 34+/Lin- cells measured in limiting dilution assays (LDA) on AFT024 (4.45 +/- 0.69%) was significantly higher than in M2-10B4 (1.45 +/- 0.20%) LDA. Addition of MIP-1alpha and IL-3 to AFT024 LDA increased the measured LTC-IC frequency to 6.8 +/- 0.9%. We also determined the fraction of LTC-IC that persisted after 34+/Lin cells were cultured for 5 weeks by replating progeny in the three LDA readout systems. The measured LTC-IC maintenance was significantly lower when M2-10B4 LDA (13.1 +/- 3.5%, P < 0.05) were used compared with AFT024 LDA (36.6 +/- 5.5%) or AFT024 LDA supplemented with MIP-1alpha and IL-3 (29.1 +/- 6.3%). Thus, the number of LTC-IC measured in freshly sorted 34+ cells depends on the stromal feeder used in LDA assays. Furthermore, and most important, assessment of LTC-IC expansion or maintenance may vary significantly depending on the type of stromal feeder used to enumerate LTC-IC.     

Selective cytokine gene expression in renal cell carcinoma tumor cells and tumor-infiltrating lymphocytes

The progression of tumors such as renal cell carcinoma (RCC), despite the presence of substantial lymphocytic infiltrates (TIL), suggests that the ability of the local immune response to control tumor growth is impaired. Cytokine gene expression was examined to further investigate the nature of this response. Initial studies were performed with frozen tumors using PCR-assisted mRNA amplification with cytokine-specific primers for interleukin 10 (IL-10), interleukin 2 (IL-2) and interferon-gamma (IFN-γ). IL-2 mRNA was not detected, despite the presence of T cells as defined by the expression of CD3γ mRNA. In contrast, mRNA for IFN-γ was expressed in 4/9 and for IL-10 in 5/9 tumors. To confirm this, 5 fresh tumor specimens were examined, and PCR demonstrated that IL-10 mRNA was detectable in 4/5 tumors from which RNA was isolated at the time of nephrectomy. In these experiments multiple cycles and dilutions were employed to semi-quantitate the expression of IL-10. To identify potential sources of this cytokine in the tumor bed, IL-10 mRNA expression in freshly isolated lymphocytes and tumor cells, TIL lines, cultured RCC and established RCC lines was examined. Our studies demonstrate that within the tumor TIL may be one source of IL-10. Lymphocyte-enriched populations from 4/5 tumors expressed IL-10 mRNA as did 4/6 freshly isolated tumor cell preparations. IL-10 gene expression was not detected, however, in tumor cells after one passage in vitro in short-term cultured RCC tumor cells (passages 2–5) or in established RCC tumor cell lines. Finally, 4/9 CD4⁺ and 2/5 CD8⁺ TIL lines expressed IL-10 mRNA either constitutively or after stimulation with anti-CD3 antibody. This finding was associated with IL-10 production in vitro. Our studies demonstrate that IL-10 mRNA is frequently present in RCC tumors and may originate from the tumor-infiltrating mononuclear cell population. © 1995 Wiley-Liss, Inc.     

The in vivo expression of type B CD23 mRNA in B-chronic lymphocytic leukemic cells is associated with an abnormally low CD23 upregulation by IL-4: comparison with their normal cellular counterparts.

We recently reported that the sera of chronic lymphocytic leukemia (CLL) patients contained 3-500 times more soluble CD23 (or IgE-BF) than the sera of patients with other lymphoproliferative diseases or normal individuals and that their B cells (B-CLLs) overexpressed CD23 Ag. In the present report, we extended these studies and showed that CD5+ B cells from all CLL patients (n = 15) co-express CD23 Ag. We next identified two additional major differences between B-CLLs and normal adult B cells. First, in contrast to normal adult B cells which exclusively express type A CD23 mRNA, freshly isolated B-CLLs expressed both type B and type A CD23 mRNA. Second, although IL-4 is a potent inducer of type B CD23 mRNA on normal B cells, an optimal concentration of IL-4 infranormally upregulated CD23 on highly purified B-CLLs both at the protein and at the molecular levels. However, co-stimulation of CLL PBMC with phytohemagglutinin (PHA) and IL-4 strongly upregulated CD23 on B-CLLs, reconstituting the high level of CD23 expression observed in vivo. We next attempted to relate B-CLLs to the CD5+ B cell subpopulations present in peripheral blood mononuclear cells (PBMC, n = 3), cord blood mononuclear cells (CBMC, n = 6) and tonsillar lymphocytes (TONS, n = 3) by analysing their co-expression of CD20, CD5 and CD23 Ag and their phenotypic regulation by IL-4. Our results indicated that B-CLLs presented some features in common with the CD23+ umbilical cord blood B cells in as much as, like in B-CLLs; (i) all CD23+ cord blood cells co-expressed CD5 Ag, (ii) freshly isolated CBMC expressed both type A and type B CD23 mRNA, and finally (iii) these cells weakly re-expressed CD23 Ag upon IL-4 stimulation as compared to adult PBMC.