CD81 but Not CD82 Dendritic Cells Cross-prime Cytotoxic T Cells In Vivo By Joke M.M. den Haan, Sophie M. Lehar, and Michael J. Bevan From the Department of Immunology, Howard Hughes Medical Institute, University of Washington, Seattle,Washington 98195 Abstract Bone marrow–derived antigen-presenting cells (APCs) take up cell-associated antigens and present them in the context of major histocompatibility complex (MHC) class I molecules to CD81 T cells in a process referred to as cross-priming. Cross-priming is essential for the induc- tion of CD81 T cell responses directed towards antigens not expressed in professional APCs. Although in vitro experiments have shown that dendritic cells (DCs) and macrophages are ca- pable of presenting exogenous antigens in association with MHC class I, the cross-presenting cell in vivo has not been identified. We have isolated splenic DCs after in vivo priming with ovalbumin-loaded b2-microglobulin–deficient splenocytes and show that they indeed present cell-associated antigens in the context of MHC class I molecules. This process is transporter as- sociated with antigen presentation (TAP) dependent, suggesting an endosome to cytosol trans- port. To determine whether a specific subset of splenic DCs is involved in this cross-presenta- tion, we negatively and positively selected for CD82 and CD81 DCs. Only the CD81, and not the CD82, DC subset demonstrates cross-priming ability. FACS® studies after injection of sple- nocytes loaded with fluorescent beads showed that 1 and 0.6% of the CD81 and the CD82 DC subsets, respectively, had one or more associated beads. These results indicate that CD81 DCs play an important role in the generation of cytotoxic T lymphocyte responses specific for cell- associated antigens. Key words: major histocompatibility complex class I • antigen presentation • antigen-presenting cell • cytotoxic T lymphocyte • cross-priming Introduction Naive CD81 T cells are stimulated to proliferate and to de- velop into cytotoxic effector T cells after recognition of short peptides associated with MHC class I molecules on professional APCs. “Endogenous” cytosolic proteins are generally the source of MHC class I–restricted antigens. These proteins are degraded by the proteasome, and pep- tides are transported to the endoplasmic reticulum by the transporter associated with antigen presentation (TAP)1 transporter, where they can bind to newly synthesized MHC class I molecules. In contrast, membrane-associated proteins and endocytosed “exogenous” proteins are mainly presented by MHC class II molecules for recognition by CD41 T cells. However, the division of the endogenous Address correspondence to Michael J. Bevan, Department of Immunol- ogy, Howard Hughes Medical Institute, University of Washington, Box 357370, Seattle, WA 98195. Phone: 206-685-3610; Fax: 206-685-3612; E-mail: mbevan@u.washington.edu 1Abbreviations used in this paper: B6, C57BL/6; b2m, b2-microglobulin; CFSE, carboxyfluorescein diacetate succinimidyl ester; DC, dendritic cell; PALS, periarteriolar lymphatic sheaths; TAP, transporter associated with antigen presentation. and exogenous pathway is not absolute. 25 years ago, it was recognized that immunization with cells lacking host MHC alleles, but bearing foreign minor histocompatibility anti- gens, leads to minor specific CD81 T cell activation that is restricted to the host MHC allele (1, 2). This suggested that host APCs can process exogenous cell-associated antigens and present them in the context of MHC class I molecules, a process termed cross-priming or cross-presentation. Since then, cross-priming has been shown to be important in ini- tiating MHC class I–restricted responses to tumors, periph- eral self, viral, and bacterial antigens (3–6). This indicates that cross-presentation is a general mechanism for the in- duction of T cells specific for antigens not expressed by APCs themselves and is involved in a variety of T cell re- sponses (7). In addition to T cell activation, cross-presenta- tion has been shown to induce T cell tolerance (8, 9). Bone marrow chimera studies demonstrated that the ability to cross-present cell-associated antigens is restricted to bone marrow–derived APCs (4, 10). Several groups have investigated the role of macrophages and dendritic cells (DCs) in presenting exogenously derived antigens in 1685 J. Exp. Med. The Rockefeller University Press • 0022-1007/2000/12/1685/11 $5.00 Volume 192, Number 12, December 18, 2000 1685–1695 http://www.jem.org/cgi/content/full/192/12/1685