Santamaria P, Lewis C, Barbosa J J
Department of Medicine, University of Minnesota, Minneapolis.
Eur J Immunol. 1993 Nov;23(11):2972-9. doi: 10.1002/eji.1830231137.
Functional and molecular studies of in vivo-activated T lymphocytes involved in normal and abnormal immune responses, i.e., cells infiltrating tissues affected by autoimmune processes, require their previous in vitro expansion. Problems such as unavailability of specific antigen(s) (Ag) and/or the requirement of large amounts of autologous peripheral blood mononuclear cells (PBMC) as feeders have prompted the development of alternative expansion methods that circumvent the use of antigen-presenting cells (APC) and/or Ag. We have previously shown that cytomegalovirus (CMV)-specific T cell lines and clones can be efficiently propagated in long-term cultures by stimulation with agonistic monoclonal antibodies (mAb) coated onto polystyrene particles in the absence of APC. However, this and other stimulation protocols may skew the repertoire of clonotypes that proliferate in response to nominal Ag and APC. Here we show that polyclonal populations of CMV-primed and interleukin-2 (IL-2)-stimulated PBMC undergo the same clonotypic selection when induced to grow both by continuous stimulation with CMV and an anti-CD3 mAb presented by APC. This selection, reproduced in three independent expansion experiments, involved the dominant growth of two CMV-specific, IL-2-secreting CD4+ clonotypes sharing J alpha, J beta, V alpha and V beta genes and highly homologous T cell receptor (TcR) junctional sequences. The dominant growth of these 2 clonotypes required a direct T cell/APC interaction since when coated onto polystyrene particles the same mAb induced the selective expansion of another IL-2-secreting CD4+ CMV-specific clonotype expressing a different, yet homologous, TcR heterodimer (used same V alpha and V beta genes), which was underrepresented before expansion (5 vs. 58%). T cell clones belonging to the subdominant clonotype proliferated significantly faster in response to stimulation with anti-CD3 mAb coated onto beads than in response to stimulation with CMV or anti-CD3 mAb presented by APC, possibly due to long-term expansion without APC or antigen. In contrast, neither the dominant clonotypes nor unprimed T cells were able to undergo CD3-mediated expansion in the absence of APC. We conclude that quantitative differences in growth competency among normal Ag-activated T-helper (Th) clonotypes in response to antigenic stimulation can be reproduced by stimulation through the TcR in the absence of TcR occupancy but only in the presence of APC and that certain clonotypes do not require APC for long-term growth in vitro. These data have practical implications for the isolation and repertoire characterization of in vivo-activated T cells from tissues affected by inflammatory, i.e. autoimmune, phenomena.
参与正常和异常免疫反应的体内活化T淋巴细胞的功能和分子研究，即浸润受自身免疫过程影响组织的细胞，需要事先在体外进行扩增。诸如特定抗原（Ag）不可用和/或需要大量自体外周血单个核细胞（PBMC）作为饲养细胞等问题，促使人们开发替代扩增方法，以规避使用抗原呈递细胞（APC）和/或Ag。我们先前已表明，巨细胞病毒（CMV）特异性T细胞系和克隆可通过在无APC的情况下用包被在聚苯乙烯颗粒上的激动性单克隆抗体（mAb）刺激，在长期培养中有效增殖。然而，这种及其他刺激方案可能会使响应名义Ag和APC而增殖的克隆型库发生偏差。在此我们表明，CMV致敏且白细胞介素-2（IL-2）刺激的PBMC多克隆群体，当通过CMV和APC呈递的抗CD3 mAb持续刺激诱导生长时，会经历相同的克隆型选择。这种选择在三个独立的扩增实验中得到重现，涉及两种CMV特异性、分泌IL-2的CD4 +克隆型的优势生长，它们共享Jα、Jβ、Vα和Vβ基因以及高度同源的T细胞受体（TcR）连接序列。这两种克隆型的优势生长需要直接的T细胞/APC相互作用，因为当包被在聚苯乙烯颗粒上时，相同的mAb诱导了另一种分泌IL-2的CD4 + CMV特异性克隆型的选择性扩增，该克隆型表达不同但同源的TcR异二聚体（使用相同的Vα和Vβ基因），在扩增前其比例较低（5%对58%）。属于次要克隆型的T细胞克隆，对包被在珠子上的抗CD3 mAb刺激的反应，比对CMV或APC呈递的抗CD3 mAb刺激的反应显著更快，这可能是由于在无APC或抗原的情况下长期扩增所致。相反，在无APC的情况下，优势克隆型和未致敏的T细胞均不能进行CD3介导的扩增。我们得出结论，正常Ag活化的T辅助（Th）克隆型在响应抗原刺激时生长能力的定量差异，可通过在无TcR占据但仅在有APC存在的情况下通过TcR刺激来重现，并且某些克隆型在体外长期生长不需要APC。这些数据对于从受炎症即自身免疫现象影响的组织中分离体内活化T细胞及其库特征分析具有实际意义。
