Dunbar P R, Ogg G S, Chen J, Rust N, van der Bruggen P, Cerundolo V
Molecular Immunology Group, Institute of Molecular Medicine, Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital Oxford, OX3 9DS, UK.
Curr Biol. 1998 Mar 26;8(7):413-6. doi: 10.1016/s0960-9822(98)70161-7.
Cytotoxic T lymphocytes (CTLs) play an important role in controlling viral infections and certain tumours, but characterising specific CTL responses has always been technically limited. Fluorogenic 'tetramers' of major histocompatibility complex (MHC) class I complexes have been exploited recently to quantify the massive expansion of specific CTLs in human immunodeficiency virus (HIV) infection [1]. Here, we use MHC class I complex tetramers to isolate low-frequency antigen-specific CTLs directly from human peripheral blood, allowing the simultaneous phenotypic and functional characterisation and cloning of these CTLs. We synthesised a tetramer that specifically stained human leukocyte antigen (HLA)-A2. 1-restricted CTL clones recognising the influenza matrix protein peptide 58-66, matrix 58-66 [2]. This tetramer stained between 1 in 1,500 and 1 in 58,000 peripheral blood mononuclear cells (PBMCs) from HLA-A2.1+ individuals. The surface phenotype of these cells could be analysed by fluorescence-activated cell sorting (FACS), and the cells could be directly sorted into enzyme-linked immunospot (ELISpot) plates, where they released interferon-gamma (IFN-gamma) within 1 day of antigen exposure. The same population was cloned by FACS, and the specificity of several expanded clones was confirmed. Cloning was greatly simplified and accelerated compared with standard protocols, and was highly efficient. We also used tetramer-based sorting to enrich melanoma-specific CTLs derived from a tumour-infiltrated lymph node. Direct cloning of specific CTLs from peripheral blood can provide important information about immunological memory, CTL responses against tumour antigens and CTL proliferation and function, and opens up new possibilities for generating CTLs for adoptive immunotherapy.
细胞毒性T淋巴细胞（CTLs）在控制病毒感染和某些肿瘤方面发挥着重要作用，但对特定CTL反应的表征在技术上一直存在局限。主要组织相容性复合体（MHC）I类复合体的荧光“四聚体”最近已被用于量化人类免疫缺陷病毒（HIV）感染中特定CTL的大量扩增[1]。在此，我们使用MHC I类复合体四聚体直接从人外周血中分离低频抗原特异性CTL，从而能够同时对这些CTL进行表型和功能表征及克隆。我们合成了一种四聚体，它能特异性地标记识别流感病毒基质蛋白肽58 - 66（基质58 - 66）的人类白细胞抗原（HLA）- A2.1限制性CTL克隆[2]。该四聚体在来自HLA - A2.1 +个体的外周血单个核细胞（PBMCs）中标记的细胞比例为1/1500至1/58000。这些细胞的表面表型可通过荧光激活细胞分选（FACS）进行分析，并且这些细胞可直接分选到酶联免疫斑点（ELISpot）板中，在暴露于抗原后1天内它们会释放干扰素 - γ（IFN - γ）。通过FACS对同一群体进行克隆，并确认了几个扩增克隆的特异性。与标准方案相比，克隆过程大大简化且加速，并且效率很高。我们还使用基于四聚体的分选方法富集来自肿瘤浸润淋巴结的黑色素瘤特异性CTL。从外周血中直接克隆特异性CTL可以提供有关免疫记忆、针对肿瘤抗原的CTL反应以及CTL增殖和功能的重要信息，并为生成用于过继性免疫治疗的CTL开辟了新的可能性。
