Cutting Edge: Characterization of Allorestricted and Peptide-Selective Alloreactive T Cells Using HLA- Tetramer Selection 1 Arnaud Moris, 2 * Volker Teichgra ¨ber,* Laurent Gauthier, ² Hans-Jo ¨rg Bu ¨hring, § and Hans-Georg Rammensee 3 * The vast majority of alloreactive T cells recognize foreign MHC molecules in a peptide-dependent manner. A subpopu- lation of these peptide-dependent alloreactive T cells is pep- tide-specific and contains T cells that are of interest for tumor immunotherapy. Allorestricted T cells (i.e., peptide-specific and alloreactive) specific for tumor-associated Ags can be raised in vitro. However, it is technically difficult to distinguish between peptide-specific and peptide-nonspecific alloreactive T cells by functional assays in vitro. Here we show for the first time that allorestricted T cells specifically bind HLA-peptide tetrameric complexes, as nominal Ag-specific T cells would do. In consequence, fluorescent HLA-peptide tetrameric com- plexes can be used for sorting and cloning of allorestricted CTLs specific for a peptide of interest. We also show by the mean of HLA-peptide tetramers the existence of peptide-selec- tive alloreactive T cells that recognize a conformation on the foreign-MHC brought about by some but not all peptides bound. The Journal of Immunology, 2001, 166: 4818 – 4821. A llograft rejection and graft-vs-host disease (GVHD) 4 are the clinical manifestations of T cell reactivity to foreign MHC molecules (1). The molecular basis of allorecog- nition has been extensively studied (2, 3). The nature of the de- terminants involved in the alloreactive T cell recognition appears to be very diverse (2). The targets of alloreactivity, the MHC class I molecules, bind 8- to 10-aa peptides from intracellular sources and display them at the cell surface. CTL clones that seem to recognize allogeneic molecules in a peptide-independent fashion have been reported (2, 4, 5). Several studies have indicated the existence of peptide-dependent but not peptide-specific CTLs (2, 6). This fraction of alloreactive T cells might be sensitive to the conformation of the MHC that is adapted when particular, but unrelated, peptides are bound (3). The existence of peptide-specific alloreactive T cells has been clearly demonstrated (2, 7–10). These allorestricted CTLs can recognize specific peptide-MHC com- plexes just like nominal Ag-specific T cells do (11–13). By using peptide libraries, our group has recently shown that the mouse as well as the human allorestricted T cell repertoire is broad and diverse (11, 13). Many tumors overexpress normal proteins thereby modifying the set of self-peptides associated with MHC class I molecules. This phenomenon allows triggering of tumor-specific CTLs. How- ever, CTLs undergo negative selection and peripheral tolerance mechanisms that diminish the number or eliminate self-peptide- specific CTLs. This is an obvious limitation to generate in vitro tumor-specific cytotoxic T cells to be used in adoptive immuno- therapy. The existence of the allorestricted repertoire raises the possibility of generating CTLs reactive against synthetic self-pep- tides bound to nonself-MHC molecules, because tolerance to self- Ags is self-MHC restricted (14). Thus, it should be possible to produce in vitro CTL against self-Ags that are expressed in tumor cells for adoptive immunotherapy (14, 15). Indeed, it has been shown recently that allorestricted CTLs specific for mdm-2 wild- type peptide can be a successful reagent for immunotherapy in mice (12). These CTLs can engraft and retain specificity in the host without causing GVHD (16). We and others (15) evaluate the pos- sibility to isolate allorestricted CTLs that originate from HLA- A*02-negative donors and recognize specifically HLA-A2-peptide complexes. However, the in vitro generation of such allorestricted T cells remains problematic because of the difficulty to separate between the large pool of alloreactive (i.e., recognizing foreign MHC) and the small fraction of allorestricted (i.e., restricted for a particular peptide on foreign MHC) CTL activities. Here, we in- vestigated whether allorestricted T cells can bind HLA-tetrameric complexes specifically, as Ag-specific T cells would do, and whether HLA tetramers can be used for sorting and cloning of allorestricted CTLs specific against a peptide of interest. *Institute for Cell Biology, Department of Immunology, University of Tu ¨bingen, Tu ¨ bingen, Germany; ² Immunotech, Beckmann-Coulter, Marseille, France; and § Med- ical Clinic, Department II, Tu ¨bingen, Germany Received for publication November 30, 2000. Accepted for publication February 26, 2001. 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 work was supported by the University of Tu ¨ bingen, “fortune program” number 406-0-0, and by the European Union grant on “CTLs follow up and vaccination: B104-98-0214.” 2 Current address: Pasteur Institute, Retrovirus and Gene Transfer, 28 rue du Dr. Roux, F-75724 Paris Cedex 15, France. 3 Address correspondence and reprint requests to Dr. Hans-Georg Rammensee, In- stitute for Cell Biology, Department of Immunology, University of Tu ¨ bingen, Auf der Morgenstelle 15, D-72076 Tu ¨bingen, Germany. E-mail address: rammensee@uni- tuebingen.de 4 Abbreviations used in this paper: GVHD, graft-vs-host disease; CEA, carcinoem- bryonic Ag; LCL, lymphoblastoid B cell line; MP, matrix protein; TyrA, tyrosinase A; GVL, graft-vs-leukemia. Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 ● ●