Santamaria P, Bryan M K, Barbosa J
Department of Medicine, University of Minnesota, Minneapolis 55455.
J Immunol Methods. 1990 Aug 28;132(1):1-11. doi: 10.1016/0022-1759(90)90392-9.
Functional and molecular studies of 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) and/or the requirement of large amounts of autologous peripheral blood mononuclear cells (PBMNCs) as feeder cells, demand the development of alternative expansion methods. Cytomegalovirus (CMV)-primed PBMNCs from several seropositive subjects were expanded for 5-6 weeks by stimulation with anti-CD3 coated onto polystyrene beads plus interleukin-2 (IL-2) with a similar efficiency than when the MAb was presented by autologous MNCs. Beads coated with anti-CD4, but not with anti-CD8, were also able to maintain the long term growth of CMV-primed populations of T cells. The expanded T cells of one of these polyclonal populations were cloned by limiting dilution using anti-CD3 or CMV, and autologous PBMNCs as stimuli. 16 and 11 clones, respectively, were obtained and grown to several million cells for 1-2 months by weekly stimulations with anti-CD3-coated beads and IL-2. Proliferation assays performed with most of the clones generated with anti-CD3 stimulation showed that all the tested clones had retained the CMV and the class II MHC restriction specificities for at least 3-4 months after the initial CMV stimulation. All the tested clones secreted IL-2 in response to CMV and were CD3+, CD4+ and CD8-. Comparison of the growth of two of these clones by stimulation with: (a) anti-CD3-coated beads and IL-2; (b) anti-CD3, autologous MNCs and IL-2; or (c) CMV, autologous MNCs and IL-2, showed that the first combination was at least as efficient as the other two in expanding these T cell clones. We conclude that polystyrene monosized particles coated with agonistic antibodies can induce the long term growth of antigen-specific T cell lines in the absence of specific antigen and feeder cells, often unavailable specially in the context of human autoimmune studies.
对参与正常和异常免疫反应的T淋巴细胞进行功能和分子研究，即对浸润受自身免疫过程影响组织的细胞进行研究，需要事先在体外进行扩增。诸如缺乏特异性抗原和/或需要大量自体外周血单个核细胞（PBMNC）作为饲养细胞等问题，要求开发替代的扩增方法。来自数名血清反应阳性受试者的巨细胞病毒（CMV）致敏的PBMNC，通过用包被在聚苯乙烯珠上的抗CD3加白细胞介素-2（IL-2）刺激，扩增5至6周，其效率与用自体MNC呈递单克隆抗体（MAb）时相似。包被抗CD4而非抗CD8的珠子，也能够维持CMV致敏的T细胞群体的长期生长。这些多克隆群体之一的扩增T细胞，通过有限稀释法，使用抗CD3或CMV以及自体PBMNC作为刺激物进行克隆。分别获得了16个和11个克隆，并通过每周用包被抗CD3的珠子和IL-2刺激，使其生长至数百万个细胞，持续1至2个月。对大多数由抗CD3刺激产生的克隆进行的增殖试验表明，所有测试克隆在最初的CMV刺激后至少3至4个月内，都保留了CMV和II类MHC限制性特异性。所有测试克隆在受到CMV刺激时都会分泌IL-2，并且是CD3 +、CD4 +和CD8 -。通过以下刺激比较其中两个克隆的生长情况：（a）包被抗CD3的珠子和IL-2；（b）抗CD3、自体MNC和IL-2；或（c）CMV、自体MNC和IL-2，结果表明第一种组合在扩增这些T细胞克隆方面至少与其他两种组合一样有效。我们得出结论，包被激动性抗体的聚苯乙烯单尺寸颗粒，可以在缺乏特异性抗原和饲养细胞的情况下，诱导抗原特异性T细胞系的长期生长，而在人类自身免疫研究中，特异性抗原和饲养细胞通常难以获得。
