Mannering S I, McKenzie J L, Hart D N
Haematology/Immunology/Transfusion Medicine Research Group, Christchurch School of Medicine, New Zealand.
J Immunol Methods. 1998 Oct 1;219(1-2):69-83. doi: 10.1016/s0022-1759(98)00125-2.
Naive T lymphocytes specific for a given primary antigen occur in low frequencies and require the relevant antigen to be presented by specialist antigen presenting cells (APC), i.e., dendritic cells (DC). For these reasons, the in vitro induction of primary T lymphocyte responses remains a significant technical challenge. We have attempted to improve current strategies for generating in vitro responses by optimising (i) isolation and concomitant activation of DC from peripheral blood, (ii) uptake, processing and presentation of antigen by DC and (iii) antigen driven T lymphocyte proliferation. We established that RPMI 1640 media supplemented with 10% autologous serum resulted in the best yield of CMRF-44+, CD14-, CD19- DC after enrichment over a Nycodenz gradient. Optimal presentation of whole protein and peptide antigen was achieved by addition after the purification of the APC, i.e., at the start of the T lymphocyte proliferation assay. RPMI 1640 supplemented with 10% autologous serum or plasma supported the best antigen driven specific T lymphocyte responses. Using these optimised conditions, we compared the efficacy of PBMC and purified blood DC for priming T lymphocyte responses to the chronic myeloid leukaemia (CML) specific bcr-abl (b3a2) peptide. Peptide specific T lymphocyte responses were generated with both purified DC and whole PBMC, suggesting that T lymphocyte precursor frequency was the limiting factor in these experiments. These results will aid in the generation of human T lymphocyte lines to primary antigens, for in vitro and therapeutic applications.
针对特定初始抗原的初始T淋巴细胞出现频率较低，并且需要由专职抗原呈递细胞（APC），即树突状细胞（DC）呈递相关抗原。由于这些原因，体外诱导初始T淋巴细胞反应仍然是一项重大的技术挑战。我们试图通过优化以下方面来改进当前产生体外反应的策略：（i）从外周血中分离并同时激活DC；（ii）DC对抗原的摄取、加工和呈递；（iii）抗原驱动的T淋巴细胞增殖。我们确定，在通过Nycodenz梯度富集后，添加10%自体血清的RPMI 1640培养基可产生最高产量的CMRF-44+、CD14-、CD19-DC。通过在APC纯化后添加，即在T淋巴细胞增殖试验开始时添加，可实现全蛋白和肽抗原的最佳呈递。添加10%自体血清或血浆的RPMI 1640支持最佳的抗原驱动特异性T淋巴细胞反应。使用这些优化条件，我们比较了PBMC和纯化的血液DC引发T淋巴细胞对慢性髓性白血病（CML）特异性bcr-abl（b3a2）肽反应的效果。纯化的DC和全PBMC均可产生肽特异性T淋巴细胞反应，这表明T淋巴细胞前体频率是这些实验中的限制因素。这些结果将有助于生成针对初始抗原的人T淋巴细胞系，用于体外和治疗应用。
