Johannisson A, Festin R
Department of Pathology, Swedish University of Agricultural Sciences, Uppsala.
Cytometry. 1995 Apr 1;19(4):343-52. doi: 10.1002/cyto.990190409.
An investigation of proliferation and activation events in subsets of human CD4+ cells, defined by their expression of CD45RA and CD45R0, is reported. A single-laser based assay for the study of multiple surface antigens and two-parameter cell cycle analysis was used for sorting of and subsequent analysis of proliferation in CD4+CD45RA+ CD45R0-, CD4+CD45RA-CD45R0+ subsets and phenotypically intermediate stages. After labelling with BrdUrd, cells were sorted with flow cytometry on the basis of light-scattering properties and staining with anti-CD45RA, anti-CD45R0, and anti-CD4 markers. Sorted cells were double stained with anti-BrdUrd-antibodies and PI, and the frequencies of proliferating cells were determined. After 48 h, the highest rate of proliferation was found among cells with a phenotype intermediate between CD4+ CD45RA+CD45R0- and CD4+CD45RA-CD45R0+. After 72 h of culture, the situation was changed insofar as the point of highest proliferation had shifted towards the CD4+CD45RA-CD45R0+ population. These findings were further corroborated by four-colour staining with anti-CD4, anti-CD45RA, anti-CD45R0, and Hoechst 33342. This indicates that the phenotype transition is accompanied by cell proliferation. The correlated temporal expression of antigens related to activation (HLA-DR, CD25, CD69, CD71) and cell adhesion (CD11a, CD54, L-selectin) in each of the different subsets was also investigated. All the activation markers CD25, CD69, and CD71 show a more heterogeneous pattern of expression among the CD4+ CD45RA-CD45R0+ cells than the CD4+ CD45RA+CD45R0- cells, indicating a subpopulation of CD4+CD45RA-CD45R0+ cells responding more slowly to the mitogenic stimulation.
本文报道了一项针对人类CD4+细胞亚群增殖和激活事件的研究，这些亚群由其CD45RA和CD45R0的表达所定义。采用基于单激光的多表面抗原研究检测方法和双参数细胞周期分析，对CD4+CD45RA+CD45R0-、CD4+CD45RA-CD45R0+亚群以及表型中间阶段的细胞进行分选和随后的增殖分析。用BrdUrd标记后，根据光散射特性以及抗CD45RA、抗CD45R0和抗CD4标记染色，通过流式细胞术对细胞进行分选。分选后的细胞用抗BrdUrd抗体和PI进行双重染色，并确定增殖细胞的频率。48小时后，在具有介于CD4+CD45RA+CD45R0-和CD4+CD45RA-CD45R0+之间表型的细胞中发现了最高的增殖率。培养72小时后，情况发生了变化，此时最高增殖点已向CD4+CD45RA-CD45R0+群体转移。用抗CD4、抗CD45RA、抗CD45R0和Hoechst 33342进行四色染色进一步证实了这些发现。这表明表型转变伴随着细胞增殖。还研究了每个不同亚群中与激活相关的抗原（HLA-DR、CD25、CD69、CD71）和细胞黏附（CD11a、CD54、L-选择素）的相关时间表达。所有激活标记CD25、CD69和CD71在CD4+CD45RA-CD45R0+细胞中的表达模式比CD4+CD45RA+CD45R0-细胞更为异质性，表明CD4+CD45RA-CD45R0+细胞亚群对有丝分裂刺激的反应较慢。
