A chaperone-assisted high yield system for the production of HLA-DR4 tetramers in insect cells Jean-Marie Fourneau, He ´le `ne Cohen, Peter M. van Endert * Institut National de la Sante ´ et de la Recherche Me ´dicale Unite ´ 580, Ho ˆpital Necker, 161 rue de Se `vres, 75015 Paris, France Received 19 August 2003; received in revised form 6 November 2003; accepted 20 November 2003 Abstract MHC tetramers have become essential tools for the analysis of antigen specific responses of CD8 + and CD4 + T cells. However, the use of MHC class II tetramers is hampered by the relatively low yields of most current expression systems. We have devised an insect cell/baculovirus expression system in which yields of 50 – 70 mg of recombinant HLA-DR4 molecules, with or without covalently linked peptide, per liter of insect cell supernatant, are routinely obtained. These yields are rendered possible by an optimized design and use of DRa and DRh expression cassettes and by co-expression of a housekeeping chaperone of the endoplasmic reticulum, calreticulin, which, due to its co-secretion, increases secretion of HLA-DR molecules two- to threefold. A tetramer produced in the system specifically was shown to stain an HLA-DR4 restricted T cell line obtained from a healthy donor by in vitro priming, but which recognizes a type I diabetes autoantigen. Co-expression of chaperones may represent a general strategy for enhancing yields of recombinant proteins expressed in insect cells and facilitate production of MHC class II tetramers in the future. D 2004 Elsevier B.V. All rights reserved. Keywords: Autoimmunity; Antigens/peptides/epitopes; MHC; Antigen presentation/processing; Molecular biology 1. Introduction The description of HLA-A*0201 tetramers (Alt- man et al., 1996) opened a new era in the study of antigen-specific T cell responses. Multimerization of MHC molecules increases the avidity of the MHC/ peptide–TCR interaction to a level where such reagents can be used in an antibody-like fashion to stain T cells recognizing a specific peptide/MHC complex. Tetramers can be used for flow cytometric staining and sorting of viable T lymphocytes as well as for immunohistochemical tracking of specific T cells (Klenerman et al., 2002); tetramers may also have potential as tools for immunotherapy (Xu and Screaton, 2002). While MHC class I tetramers have allowed tre- mendous advances in our understanding of CTL responses to viruses and tumors, and of homeostatic control of T cell populations, insights resulting from the use of class II tetramers have so far been much more limited. This is partly due to—relative to CD8 + cells—the much lower frequencies of CD4 + cells specific for a given antigen; in addition, production of class II tetramers has been technically more chal- 0022-1759/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2003.11.011 Abbreviations: ER, endoplasmic reticulum; MCS, multiple cloning site; BCA, bicinchronic acid; GAD65, 65-kDa glutamic acid decarboxylase. * Corresponding author. Tel.: +33-144492563; fax: +33- 144495382. E-mail address: vanendert@necker.fr (P.M. van Endert). www.elsevier.com/locate/jim Journal of Immunological Methods 285 (2004) 253 – 264