Antigenspecific Cells Enrichment And Isolation

KGC Smith, Department of Medicine, University of Cambridge, School of Clinical Medicine, Addenbrooke's Hospital, Cambridge, UK

JE Layton, Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Post Office Royal Melbourne Hospital, Victoria, Australia

DM Tarlinton, The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria, Australia

Burnet, in his clonal selection theory of antibody formation, proposed that the precursors of antibody-forming cells carried receptors of unique specificity for a particular antigen. After 'selection' by antigen, the antigen-specific cell would proliferate and eventually give rise to a clone of antibody-secreting cells. The antibody produced would be specific for the original antigen. Given the large number of different antigens in the environment, for any single antigen the frequency of antigen-binding cells would have to be very low, and an individual antigen-binding cell should bind only one antigen. The validity of the clonal selection theory was first demonstrated for B lymphocytes, but was subsequently shown to be true for T lymphocytes, which are also antigen specific.

Antigen-binding cells were first demonstrated and enumerated with large particulate antigens such as bacteria and red blood cells. Binding of soluble antigen by lymphocytes was first visualized by Naor and Sulitzeanu, who used ,25I-labeled albumin and autoradiography. The first antigen-binding lymphocytes studied were B cells, which recognize antigen with a unique antigen receptor comprising surface immunoglobulin. The fact that B cells bear this antigen receptor which recognizes antigenic epitopes in their native form has made isolation of antigen-specific B cells relatively successful. In contrast, T cells recognize antigen after processing to peptide form and in the context of major histocompatibility complex (MHC) molecules, and do not bind native antigen. This has made their isolation considerably more difficult. Problems with the isolation of antigen-specific cells are compounded by the relative scarcity of these cells. Even at the height of the B cell response to haptens such as (4-hydroxy-3-nitrophenyl)acetyl (NP) in the mouse, the antigen-specific B cells constitute no more than 1% spleen cells, declining to <0.01% of cells in the memory cell population. The other problem is that antigen-specific lymphocytes may differ only subtly from their colleagues, and detection of this subtle difference is critical to their isolation.

Early attempts to deplete antigen-specific cells were made to provide evidence in support of the clonal selection theory. These techniques are no longer in common use and will be considered only briefly. Detailed study of the immune response to both experimental immunogens and to pathogens is greatly enhanced by the isolation of antigen-specific lymphocytes. Techniques for such enrichment and isolation are more advanced for B cells, which will be considered first. Recent techniques employed for the isolation of antigen-specific T cells will then be examined.

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