DIRECT ISOLATION, PHENOTYPING AND CLONING OF LOW-FREQUENCY ANTIGEN-SPECIFIC CYTOTOXIC T-LYMPHOCYTES FROM PERIPHERAL-BLOOD

Cytotoxic T lymphocytes (CTLs) play an important role in controlling v iral infections and certain tumours,but characterising specific CTL re sponses has always been technically limited, Fluorogenic 'tetramers' o f major histocompatibility complex (MHC) class I complexes have been e xploited recently to quantify the massive expansion of specific CTLs i n human immunodeficiency virus (HIV) infection [1], Here, we use MHC c lass I complex tetramers to isolate low-frequency antigen-specific CTL s directly from human peripheral blood, allowing the simultaneous phen otypic and functional characterisation and cloning of these CTLs,We sy nthesised a tetramer that specifically stained human leukocyte antigen (HLA)-A2.1-restricted CTL clones recognising the influenza matrix pro tein 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) fr om HLA-A2.1(+) individuals. The surface phenotype of these cells could be analysed by fluorescence-activated cell sorting (FACS), and the ce lls could be directly sorted into enzyme linked immunospot (ELISpot) p lates, where they released interferon-gamma (IFN-gamma) within 1 day o f antigen exposure, The same population was cloned by FACS, and the sp ecificity of several expanded clones was confirmed, Cloning was greatl y 'simplified and accelerated compared with standard protocols, and wa s highly efficient, We also used tetramer-based sorting to enrich mela noma-specific CTLs derived from a tumour-infiltrated lymph node, Direc t 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 possibil ities for generating CTLs for adoptive immunotherapy.