Design of cassette vectors permitting cloning of all types of human TCR variable α and β regions
Design of cassette vectors permitting cloning of all types of human TCR variable α and β regions
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DOI：
10.1016/s0022-1759(01)00420-3
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发表时间：
2001-09-01
影响因子：
2.2
通讯作者：
De Berardinis, P
中科院分区：
文献类型：
--
作者：
Caivano, A;D'Apice, L;De Berardinis, P
T细胞克隆是研究与人类病理学相关的免疫反应的不可替代的资产。然而，这些细胞并不总是能长期培养。为了将功能性人T细胞受体（TCR）重建成稳定且快速生长的杂交瘤T细胞，我们开发了一种基于通用盒系统的通用方法，其允许克隆与鼠恒定区融合的所有类型的人T细胞受体可变α和β区基因。这些嵌合构建体易于切除并转移到可用于转染表达人CD 4的鼠T细胞杂交瘤受体的表达载体中。所得的转染子在T细胞受体-CD 3表达和IL-2对特异性抗原刺激的应答方面都是高度稳定的。使用这些盒式载体，我们重建了两个人T细胞克隆的原始HLA限制性抗原特异性，一个识别HIV-I gp 120的免疫显性表位，另一个识别HIV-I逆转录酶的免疫显性表位。我们发现，重建的杂交瘤保持了原始T细胞克隆的能力，以识别适当的表位在相关的MHC的背景下，无论是作为合成肽或加工后。它们无限的生长能力使其特别适合体外研究。(C)2001 Elsevier Science B. V.保留所有权利。
T cell clones are an irreplaceable asset for the study of immune responses relevant to human pathologies. Such cells, however, cannot always be maintained in long-term culture. In order to reconstitute functional human T cell receptors (TCRs) into stable and fast growing hybridoma T cells, we developed a general approach based on a versatile cassette system, which allows cloning of all types of human T cell receptor variable alpha and beta region genes fused to murine constant regions. These chimeric constructs are easily excised and transferred into expression vectors that can be used to transfect a human CD4-expressing murine T cell hybridoma recipient. The resulting transfectants are highly stable both in terms of T cell receptor-CD3 expression and IL-2 response to the specific antigenic stimulus. Using these cassette vectors, we reconstituted the original HLA-restricted antigen specificity for two human T cell clones, one recognizing an immunodominant epitope of HIV-I gp 120, and the other recognizing an immunodominant epitope of HIV-I reverse transcriptase. We found that the reconstituted hybridomas maintain the ability of the original T cell clones to recognize the appropriate epitope in the context of the relevant MHC either as a synthetic peptide or after processing. Their unlimited growth capacity makes them particularly suited for in vitro studies. (C) 2001 Elsevier Science B.V. All rights reserved.