1508 zyxwvutsrqpon M. Juan, R.Vilella, J. Mila et al. Eur. J. Immunol. 1993. 23: 1508-1512 z Manel JuanA, Ramon VilellaA, Jordi MilaA, Jordi YagiieA, Agusti Miralles., Keny S. Campbelle, R. Joachim Friedricho, John Cambiero, Jordi VivesA, Antonin R. De Fougerollesmand Timothy A. Springer. Servei d’ImmunologiaA, Hospital Wc, Barcelona, Division of Basic Scienceso, Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver and Committee on Immunologym, Department of Pathology, Harvard Medical School and Center for Blood Research, Boston CDw50 and ICAM-3: Two names for the same molecule* CDwSO differentiation antigen is a molecule broadly expressed on hematopoetic cells but not on other cells. Previous experiments showed that CDwSO monoclonal antibodies (mAb) inhibited primary mixed lymphocyte culture (MLC).To understand the function of CDwSO better, we purified it and obtained peptide sequence. At the same time, intercellular adhesion molecule (1CAM)-3, the third ligand of lymphocyte function-associated molecule 1, was described by mAb and subsequent cDNA cloning. Immunochemical, functional, and protein sequencing studies show that ICAM-3 and CDwSO are the same glycoprotein, a 120-kDa surface molecule with presumably an important role in the immune responses. 1 Introduction CDw50 was one of the 23 new clusters of differentiation (CD) assigned during the Fourth International Workshop on Human Leukocyte Differentiation Antigens [l]. CDwSO was originally defined by two mAb, 101-1D2 and 140-11, both produced and characterized in one of our laboratories. The ability of the CDwSO mAb to inhibit allorecognition [2] identifies CDwSO as a relevant molecule involved in leukocyte interactions. At the same time, the study of the lymphocyte function- associated molecule 1 (LFA-l), an integrin that mediates a wide range of leukocyte interactions with other cells in immune and inflammatory responses, led to the discovery of its ligands, intercellular adhesion molecule (1CAM)-1, -2 and -3 [3-61. Anti-ICAM-3 mAb have been raised [6], and recently the molecule has been cloned [7-91. ICAM-3 was found to be a member of the immunoglobulin (1g)-like supergene family, containing five Ig-like domains that are highly homologous to those found in ICAM-1 and ICAM-2. The pattern of reactivity of the CDwSO and ICAM-3 mAb seemed to be strikingly similar with a few differences. Both studies [2, 51 indicated that CDwSO and ICAM-3 may be restricted to hematopoetic cells and that both molecules have a similar molecular mass (M,) of approximately 12& 130 k D a. [I 115331 * This work was supported by “Fondo de Investigaciones Sanita- rias de la Seguridad Social” grant 91/0166. Correspondence: Manel Juan, Servei dImmunologia, Hospital Clinic, E-08036 Barcelona, Spain Key zyxwvutsrqpo words: CDw50 / ICAM-3 / LFA-1 We now report that ICAM-3 and CDwSO are the same glycoprotein. This is demonstrated by a comparison of cell distribution, sequential immunoprecipitations, transfec- tion assays with ICAM-3 cDNA, and finally comparison between the cDNA sequence of ICAM-3 and peptide sequences of CDwSO purified by affinity chromatography. The identity of ICAM-3 as CDwSO allows for a more detailed understanding of its role in immune responses. 2 Materials and methods 2.1 Monoclonal antibodies The following murine mAb t o human antigens were used: CBR-IC3/1 (anti-ICAM-3, IgG1) [6], CBR-IC3/2 (anti- ICAM-3, IgG2a) [9], 101-1D2 (anti-CDwSO, IgG1) [2], 140-11 (anti-CDwSO, IgG2b) [2], 152-2D1l (anti-CDwSO, IgGl), Cris-1 (anti-CDS, IgG2a) [lo] and 134-2C2 (IgM, CD26) [11],W6/32(anti-HLA-A, B, C, IgG2a) [12],TS1/22 (anti-CDlla, IgG1) [13], NS1 andnonbindingcontrolmAb X63 (IgG1). 2.2 Immunofluorescence assay (IF) PBMC and cell lines (10‘YO.l ml) were washed with IF buffer (PBS containing 0.02 mM NaN3 and 1% BSA) and incubated with rnAb at saturating concentration for 30 min at 4 “C, washed twice with IF buffer, and then incubated with FITC-conjugated goat anti-mouse Ig (Sigma, St. Louis, MO) for 30 min at 4 “C. Cells were analyzed on a FACStar-plus (Becton Dickinson, Mountainview, CA). In the cross-blocking experiment with CDwSO and ICAM-3 mAb, PHA-stimulated PBMC were first incubated with saturating concentrations of each antibody, washed twice with IF buffer, and then stained with fluorescein-conju- gated CDwSO mAb. The Cris-1 (a CD5 mAb) and NS1 ascites were utilized as controls. The positive cell percen- 0014-2980/93/0707-1508$10.00 + zyxwvutsr .25/0 zyxwvutsrq 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1993