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Fluorescence-Activated Cell Sorting and Cloning of Bona Fide CD8⁺ CTL with Reversible MHC-Peptide and Antibody Fab' Conjugates¹

Philippe Guillaume^*, Petra Baumgaertner, Georgi S. Angelov^*, Daniel Speiser and Immanuel F. Luescher^2,*

^* Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland; and Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland

The isolation of subsets of Ag-specific T cells for in vitro and in vivo studies by FACS is compromised by the fact that the soluble MHC-peptide complexes and Abs used for staining, especially when combined, induce unwanted T cell activation and eventually apoptosis. This is especially a problem for CD8⁺ CTL, which are susceptible to activation-dependent cell death. In this study, we show that reversible MHC-peptide complexes (tetramers) can be prepared by conjugating MHC-peptide monomers with desthiobiotin (DTB; also called dethiobiotin) and multimerization by reaction with fluorescent streptavidin. While in the cold these reagents are stable and allow good staining, they rapidly dissociate in monomers at elevated temperatures, especially in the presence of free biotin. FACS cloning of Melan-A (MART-1)-specific CTL from a melanoma-infiltrated lymph node with reversible HLA-A2 Melan-A_26–35 multimers yielded over two times more clones than when using the conventional biotin-containing multimers. CTL clones obtained by means of reversible multimers killed Melan-A-positive tumor cells more efficiently as compared with clones obtained with the stable multimers. Among the CTL obtained with the reversible multimers, but much less among those obtained with the stable multimers, a high proportion of clones exhibited high functional and physical avidity and died upon incubation with soluble MHC-peptide complexes. Finally, we show that Fab' of an anti-CD8 Ab can be converted in reversible DTB streptavidin conjugates the same way. These DTB reagents efficiently and reversibly stained murine and human CTL without affecting their viability.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This study was supported by grants from Swiss National Foundation (no. 310000-108251) and the Swiss Cancer League (OCS 01421-08-2003).

² Address correspondence and reprint requests to Dr. Immanuel F. Luescher, Ludwig Institute for Cancer Research, Lausanne Branch, 1066 Epalinges, Switzerland. E-mail address: immanuel.luescher{at}isrec.unil.ch

³ Abbreviations used in this paper: pMHC, peptide-MHC class I; BSP, biotinylation sequence peptide; DTB, desthiobiotin (or dethiobiotin); TILN, tumor-infiltrated lymph node; ABA, 4-azido-benzoic azide; Dap, di-aminopropionic acid; 7AAD, 7-aminoactinomycin D; MFI, mean fluorescence intensity.