Bregni M, Di Nicola M, Siena S, Belli N, Milanesi M, Shammah S, Ravagnani F, Gianni A M
Cristina Gandini Transplantation Unit, Istituto Nazionale Tumori, Milan, Italy.
Haematologica. 1998 Mar;83(3):204-8.
The increased susceptibility to gene transfer by amphotropic retroviral vectors of mobilized peripheral blood (PB) CD34+ cells compared to their bone marrow (BM) counterparts may depend, among other factors, on the level of expression of the amphotropic receptor on the progenitor cell. Using a previously described flow cytometry strategy, we have studied retrovirus binding to mobilized CD+ cells, derived from cancer patients treated with high-dose chemotherapy and growth factor(s), that are efficiently transduced by N2 retrovirus vector.
We measured the binding of the retrovirus to the cells using a rat monoclonal antibody reactive with the gp70 envelope glycoprotein, common to all replication-defective amphotropic retroviruses. Antibody-virus-cell complexes were indirectly labeled and analyzed by flow cytometry. We compared the binding of PA317-N2 vector to CD34+ cells derived from steady-state BM, steady-state PB and mobilized PB from cancer patients treated with high-dose chemotherapy and cytokine.
The fluorescence intensity of mobilized CD34+ cells was approximately one log higher than that of steady-state BM or PB CD34+ cells, indicating that the expression of the amphotropic receptor was increased. Moreover, the virus binding was proportional to the gene transfer rate, as assessed by G418 resistance into mobilized PB-derived CFU-GM. The increase in fluorescence intensity appeared to be restricted to CD34+ cell subset, neither CD2+ nor CD14+ cells bound the virus in an appreciable amount.
Virus binding, as assessed by indirect immunofluorescence assay, is increased in mobilized CD34+ cells. The increased binding may contribute to their high susceptibility to retrovirus vector infection.
与骨髓（BM）来源的CD34+细胞相比，动员的外周血（PB）CD34+细胞对嗜双性逆转录病毒载体基因转移的易感性增加，这可能取决于多种因素，其中包括祖细胞上嗜双性受体的表达水平。我们采用先前描述的流式细胞术策略，研究了逆转录病毒与动员的CD+细胞的结合情况，这些细胞来自接受大剂量化疗和生长因子治疗的癌症患者，可被N2逆转录病毒载体有效转导。
我们使用一种与所有复制缺陷型嗜双性逆转录病毒共有的gp70包膜糖蛋白反应的大鼠单克隆抗体，测量逆转录病毒与细胞的结合。抗体 - 病毒 - 细胞复合物通过间接标记，并用流式细胞术进行分析。我们比较了PA317 - N2载体与来自稳态骨髓、稳态外周血以及接受大剂量化疗和细胞因子治疗的癌症患者动员外周血的CD34+细胞的结合情况。
动员的CD34+细胞的荧光强度比稳态骨髓或外周血CD34+细胞高约一个对数，表明嗜双性受体的表达增加。此外，如通过对动员的外周血来源的CFU - GM进行G418抗性评估，病毒结合与基因转移率成正比。荧光强度的增加似乎仅限于CD34+细胞亚群，CD2+和CD14+细胞均未大量结合病毒。
通过间接免疫荧光测定评估，动员的CD34+细胞中病毒结合增加。结合增加可能导致它们对逆转录病毒载体感染的高易感性。
