Human vascular endothelial cells stimulate a lower frequency of alloreactive CD8+ Pre-CTL and induce less clonal expansion than matching B lymphoblastoid cells:: Development of a novel limiting dilution analysis method based on CFSE labeling of lymphocytes
Human vascular endothelial cells stimulate a lower frequency of alloreactive CD8+ Pre-CTL and induce less clonal expansion than matching B lymphoblastoid cells:: Development of a novel limiting dilution analysis method based on CFSE labeling of lymphocytes
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DOI：
10.4049/jimmunol.166.6.3846
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发表时间：
2001-03-15
影响因子：
4.4
通讯作者：
Pober, JS
中科院分区：
文献类型：
--
作者：
Dengler, TJ;Johnson, DR;Pober, JS
我们先前已经表明，人内皮细胞（EC）的效率低于专业APC，例如，B类淋巴母细胞（BLC），刺激同种异体CD 8（+）T细胞发育成CTL。在这项研究中，我们描述了基于FAGS的有限稀释分析，使用细胞内染料CFSE的稀释度作为CD 8（+）T细胞同种异体激活和扩增的指标，与传统的基于细胞毒性的测定相比，灵敏度显著增加。此外，该测定允许确定在个体CD 8（+）T细胞培养物中产生的克隆CTL群体的相对大小（克隆爆发大小）。我们应用这种方法定量比较了同种异体CD 8（+）T细胞在BLC或HUVEC刺激后克隆水平上CTL的产生。通过同种异体刺激扩增的CD 8（+）T细胞被鉴定为CD 8（+），CFSE低细胞，并且通过细胞内穿孔素和IFN-γ的表达被分类为CTL。与BLC刺激的CTL相比，EC刺激的CTL的前体频率低5- 40倍（平均，7.5倍）（p < 0.01）。同时，EC刺激的CTL培养物中的平均克隆爆发大小显著小于常规CTL培养物中的平均克隆爆发大小，这主要是由于仅用BLC刺激时出现了一些非常大的克隆大小。尽管EC刺激的CTL仅由CD 8（+）T细胞的记忆亚群产生，从初始和记忆CD 8（+）T细胞前体中观察到BLC刺激的非常大的CTL爆发大小。这些数据表明，与专业APC相比，EC促进同种异体反应性CTL分化的能力降低，反应性前体的频率较低，克隆扩增更有限，但调节性T细胞除外。
We have previously shown that human endothelial cells (EC) are less efficient than professional APC, e.g., B lymphoblastoid cells (BLC), at stimulating allogeneic CD8(+) T cells to develop into CTL. In this study we describe FAGS-based limiting dilution analyses using the dilution of the intracellular dye CFSE as an indicator of CD8(+) T cell alloactivation and expansion with significantly increased sensitivity compared with conventional, cytotoxicity-based assays. In addition, this assay permits the relative size of clonal CTL populations that are generated in individual CD8(+) T cell cultures to be determined (clonal burst size). We have applied this method to quantitatively compare the generation of CTL at the clonal level following stimulation of allogeneic CD8(+) T cells by either BLC or HUVEC derived from the same donor. CD8(+) T cells expanded by allostimulation were identified as CD8(+), CFSElow cells and were categorized as CTL by the expression of intracellular perforin and IFN-gamma, Precursor frequencies for EC-stimulated CTL were 5- to 40-fold (mean, 7.5-fold) lower compared with BLC-stimulated CTL (p < 0.01). Concomitantly, the average clonal burst sizes in EC-stimulsited CTL cultures were significantly smaller than those in conventional CTL cultures, primarily due to the occurrence of some very large clone sizes exclusively with BLC stimulation, Although EC-stimulated CTL were generated only from the memory subset of CD8(+) T cells, BLC-stimulated very large burst sizes of CTL were observed from both naive and memory CD8(+) T cell precursors. These data establish that both a lower frequency of reactive precursors and more limited clonal expansion, but not regulatory T cells, contribute to the reduced capacity of EC to promote alloreactive CTL differentiation compared with that of professional APC.