Eur. J. Immunol. 1002. 22; 2809-2817 zyxwvutsrq Epitopes of CDS8 2809 Thomas J. Dengler, Jiirg C. Hoffmann, Percy Knolle, Marion Albert-Wolf, Matthias ROUX, Reinhard Wallich and Stefan C. Meuer Division of Applied Immunology, German Cancer Research Center, Heidelberg Structural and functional epitopes of the human adhesion receptor CD58 (LFA-3) CD58 (LFA-3), a heavily glycosylated protein of 40-70 kDa, is expressed on a broad range of hematopoietic and non-hematopoietic cells. It serves as a physiological ligand of the CD2 receptor, present on Tcells and natural killer cells. and plays, thus, an important role in lymphocyte adhesion and Tcell activation through CD2. Whereas several epitopes and their respective function are known for CD2, a similarly detailed characterization of CDS8 is still lacking. We raised a panel of novel murine monoclonal antibodies (mAb) against recombinant human CD58 and describe here the identification of six structurally and/or functionally distinct epitopes on the CD58 molecule. All epitopes were found to be present in equal numbers on a wide range of CD58+ cells, none of them being differentially up-regulated following cell activation or malignant transformation. Two of these epitopes represent functionally relevant sites, involved in binding of CD58 to CD2 and Tee11 activation via CD2. One further epitope appears to be selectivcly involved in CD58-mediated activation, whereas the other three displayed no functional effects. The new mAb allow for the first time the detection of CD58 in enzyme-linked immunosorbent assays and immunofluorescence while bound to its receptor CD2 in human serum or on freshly isolated blood cells. Finally, one mAb was found to specificallycross-react with T1 ITS, the equivalent of CD58 in sheep. 1 Introduction The role of accessory receptors in Tcell activation is now becoming increasingly well defined [l-51. One of these accessory surface molecules is the glycoprotein CD2 (T11, E-rosette receptor) zyxwvutsrq [6] which is expressed on all human non-B lymphocytes and plays a central role in antigen- dependent as well as -independent T cell activation and T cell adhesion processes zyxwvutsr [ 1, 7-1 11.Thephysiological ligand of the CD2 receptor is CD58 (lymphocyte function asso- eiated-antigen 3, LFA-3) [9, 12-141, a 40-70 kDa glycopro- tein [ 121 expressed on a wide range of hematopoietic and non-hematopoietic cells, including endothelium, epithe- lium and various stromal tissues (7, 12, 15, 161. It is a member of the immunoglobulin superfamily with two extracellular domains [ 12, 17, 181, the distal of which is thought to interact with CD2 1191, itself also a member of the Ig superfamily with approximately 70% homology to CD58 [ 171. The interaction of CD2 and CD58 is involved in many essential immunological processes: both cell-associated and soluble CDS8 can activate Tcells via an “alternative pathway” in conjunction with other mitogenic CD2 mAb 120-241. CD58 on APC provides a second signal for Tcells, thus complementing and optimizing the proliferative res- ponse to TcR/CD3-mediated stimulation [25,26]. CD2/CD58 induced signaling events are unique in causing altered It-1 and IL-6 responsiveness [27] and dephospho- rylation of a recently desribed 19-kDa phosphoprotein in Tcells (281. CDS8 is furthermore involved in antigen- [I 104911 ~~ ~ ~ Correspondence: Thomas J. Dengler, Applied Immunology, Ger- man Cancer Research Center, Im Neuenheimer Feld 280, D-6900 Heidelberg, FKG independent attachment of NK cells and CTL to their targets 17, 29, 301. The CD2/CDS8 system has been suggested to contribute to the interaction of endothelial cells (EC) and Tcells, a prerequisite for lymphocyte recirculation, T cell activation and cytokine release by EC 1311. InT-B eel1collaboration CD58 plays an important role in stabilizing intercellular contact, thus reducing the requirement for nominal antigen and increasing Ig secre- tion [32, 331. CD58 expression is enhanced by cell activation and indu- cible by a variety of cytokines [34] (J. C. Hoffmann, submitted for publication). Thus, an increased number of CD58-expressing cells is found in areas of inflammation: as an example de zyxwvu now expression of CD58 on hepatocytes has been demonstrated in acute and chronic active hepatitis [35]. Findings with T leukemic cells also suggest a role for CD58 in tumor cell surveillance and clearance [36]. Finally, the recent description of a circulating form of CD58 in human serum indicates a possible function of soluble CD58 as an immune modulator (J. C. Hoffmann, manuscript submitted for publication). In contrast to the CD2 Ag 11, 14, 371, a precise epitopic characterization both in structural and functional terms has not yet been accomplished for CD58. Here we describe the identification of six structurally and/or functionally distinct epitopes of human CDS8 by a panel of novel mAb raised against recombinant CD58. 2 Materials and methods 2.1 Reagents/mAb/lymphokines Recombinant CD58 (rCD58) in an oligomeric form was expressed in the baeulovirus system (transfer vector pACC5) and purified by affinity chromatography with mAb z 0 VCII Vcrlagsgescllschaft mbTl, D-6940 Weinheim, 1902 001%2980/Y2/1 I 11-2809$3.50 + .2S/O