Britten Cedrik M, Meyer Ralf G, Frankenberg Nadine, Huber Christoph, Wölfel Thomas
III Department of Medicine, Hematology and Oncology, Johannes Gutenberg-University of Mainz, Langenbeckstrasse 1, D-55101 Mainz, Germany.
J Immunol Methods. 2004 Apr;287(1-2):125-36. doi: 10.1016/j.jim.2004.01.026.
Most assay systems for the quantification of antigen-specific T-cell responses in infectious, malignant and autoimmune disease depend on the peptide antigen format and are therefore restricted to known epitopes and their presenting HLA molecules. Here we tested in ELISPOT assays the application of in vitro-transcribed clonal mRNA as an alternative antigen format covering all potential epitopes of a given antigen. As model antigens, we chose pp65 of human cytomegalovirus (HCMV) and human tyrosinase (hTyr). Antigen-presenting cells (APC) were K562 cells stably transfected with single HLA class I alleles and autologous dendritic cells (DC). As effectors, we applied in vitro-generated anti-tyrosinase T-cell populations as well as ex vivo-CD8(+) lymphocytes from HCMV-seropositive donors. APC electroporated with clonal mRNA were efficient inducers of spot formation by antigen-experienced CD8(+) T cells. They were equivalent to peptide-loaded targets. mRNA electroporation did not induce non-specific spot formation. While the use of autologous mRNA-electroporated DC can uncover the complete individual T-cell response towards an antigen, mRNA-electroporated K562 cells stably transfected with single HLA class I alleles help to detect CD8(+) T-cell responses restricted by single HLA class I molecules.
大多数用于量化感染性、恶性和自身免疫性疾病中抗原特异性T细胞反应的检测系统都依赖于肽抗原形式，因此仅限于已知表位及其呈递的HLA分子。在此，我们在ELISPOT检测中测试了体外转录的克隆mRNA作为一种替代抗原形式的应用，它涵盖了给定抗原的所有潜在表位。作为模型抗原，我们选择了人类巨细胞病毒（HCMV）的pp65和人类酪氨酸酶（hTyr）。抗原呈递细胞（APC）是稳定转染了单个HLA I类等位基因的K562细胞和自体树突状细胞（DC）。作为效应细胞，我们应用了体外生成的抗酪氨酸酶T细胞群体以及来自HCMV血清阳性供体的体外CD8(+)淋巴细胞。用克隆mRNA电穿孔的APC是抗原经验丰富的CD8(+) T细胞形成斑点的有效诱导剂。它们等同于负载肽的靶标。mRNA电穿孔不会诱导非特异性斑点形成。虽然使用自体mRNA电穿孔的DC可以揭示个体对抗原的完整T细胞反应，但用单个HLA I类等位基因稳定转染的mRNA电穿孔的K562细胞有助于检测受单个HLA I类分子限制的CD8(+) T细胞反应。
