Paige C J, Skarvall H, Sauter H
J Immunol. 1985 Jun;134(6):3699-704.
We established culture conditions under which fetal liver-derived B cell progenitors divide and differentiate in semisolid agar, forming colonies containing antibody-secreting cells. An important feature of this assay system is that, under certain conditions, it is limiting for only one component. This was revealed by determining the slope of the least-squares log-log regression line generated when the initial seeding density was varied. When support for the growth of the clonable pre-B cells was provided by fetal liver-derived adherent accessory cells, the slope of the regression line was 1, consistent with the interpretation that only one component, the pre-B cell, was limiting under these conditions. This interpretation was tested in the present report by positive selection for cells expressing B220, Lyb-2, or AA4.1, surface markers known to be present on cell lines of the B lineage. In all cases, an increased incidence of clonable pre-B cells was observed. Furthermore, regression analysis of titration experiments undertaken with these enriched populations revealed that the assay was still limiting for a single component. A second set of growth conditions have been defined in which the clonable pre-B assay appears to be limiting for more than one component. These conditions are obtained when the adherent accessory cells are replaced by one of three distinct hemopoietic growth factors, including CSF-1 (macrophage growth factor), GM-CSF (neutrophil/macrophage growth factor), or Multi-HGF (multi-lineage hemopoietic growth factor, also called BPA or IL 3). The most straightforward interpretation of these data is that a second cell type, distinct from the B cell precursor and dependent on the growth factors, was limiting under these conditions. In the present report, this hypothesis was confirmed because growth factor responsiveness was completely absent in B220 and Lyb-2-enriched populations. Factor responsiveness was found in unseparated fetal liver and in AA4-enriched populations. However, the AA4-enriched populations, in contrast to the B220- or Lyb-2-enriched populations, also contained a large number of factor-responsive neutrophil/macrophage colonies, raising the possibility that the effect of factors on AA4+ clonable pre-B cells was accessory cell-mediated.
我们建立了培养条件，在此条件下，源自胎儿肝脏的B细胞祖细胞在半固体琼脂中分裂并分化，形成含有抗体分泌细胞的集落。该检测系统的一个重要特征是，在某些条件下，它仅受一种成分的限制。这是通过确定当初始接种密度变化时生成的最小二乘对数-对数回归线的斜率来揭示的。当源自胎儿肝脏的贴壁辅助细胞为可克隆前B细胞的生长提供支持时，回归线的斜率为1，这与在这些条件下仅一种成分即前B细胞受到限制的解释一致。本报告通过对表达B220、Lyb-2或AA4.1的细胞进行阳性选择来检验这一解释，这些表面标志物已知存在于B谱系的细胞系上。在所有情况下，均可观察到可克隆前B细胞的发生率增加。此外，对用这些富集群体进行的滴定实验的回归分析表明，该检测仍然受单一成分的限制。已经定义了第二组生长条件，在这些条件下，可克隆前B细胞检测似乎受不止一种成分的限制。当贴壁辅助细胞被三种不同的造血生长因子之一替代时，可获得这些条件，这三种因子包括CSF-1（巨噬细胞生长因子）、GM-CSF（中性粒细胞/巨噬细胞生长因子）或Multi-HGF（多谱系造血生长因子，也称为BPA或IL-3）。对这些数据最直接的解释是，在这些条件下，一种不同于B细胞前体且依赖于生长因子的第二种细胞类型受到限制。在本报告中，这一假设得到了证实，因为在富集了B220和Lyb-2的群体中完全没有生长因子反应性。在未分离的胎儿肝脏和富集了AA4的群体中发现了因子反应性。然而，与富集了B220或Lyb-2的群体不同，富集了AA4的群体还包含大量因子反应性中性粒细胞/巨噬细胞集落，这增加了因子对AA4+可克隆前B细胞的作用是由辅助细胞介导的可能性。
