A monoclonal antibody against an activation epitope on mouse integrin chain beta 1 blocks adhesion of lymphocytes to the endothelial integrin alpha 6 beta 1.

Abstract

We have generated a monoclonal antibody (mAb), 9EG7, against mouse endothelial cells that blocks adhesion of lymphocytes to endothelial cells. Sequencing offour tryptic peptides ofthe purified antigen revealed its identity with the integrin chain (3. The only Pu integrin that is known to mediate cell-cell adhesion is a4fi . This is not the integrin that is functionally dermed by the mAb 9EG7 on endothelial cells. First Antibodies. The following mAbs against mouse integrin chains were used: PS/2 (24) (anti-a4), R1-2 (16) (anti-a4), GoH3 (25) (anti-a6), and EA-1 (26, 27) (anti-a6). Other mAbs were K20 (28) (anti-human integrin chain (31) and (anti-mouse VCAM-1). Rabbit antibodies against a peptide corresponding to the C-terminal domain of the chicken integrin chain (31 were obtained from Richard 0. Hynes (MIT).The mAb 9EG7 (IgG2a) was produced by immunizing rats with TME cells in phosphate-buffered saline (PBS), using Alu-Gel-S (Serva) as adjuvant. Hybridoma production was done essentially as described (30) except that the mouse myeloma Sp2/0 was used for the fusion. Hybridoma supernatants were screened in cell-surface ELISA assays on TME Abbreviations: CAM, cell adhesion molecule; mAb, monoclonal antibody; PMA, phorbol 12-myristate 13-acetate.lTo whom reprint requests should be addressed. 9051

In vivo CD44‐CD49d complex formation in autoimmune disease has consequences on T cell activation and apoptosis resistance

“…Whether the costimulatory activity of CD49d in autoimmune LNC is due to the CD44-CD49d association or to integrin transregulation, remains to be explored [36]. The observation that AA LNC adhesion was strikingly inhibited by an antibody blocking activated CD29 [42] supports the hypothesis. The recruitment of CD49d into GEM could also strengthen b1 integrin transactivation.…”

“…AA LNC adhered more efficiently to HA than control LNC. HA adhesion of AA LNC was blocked by anti-CD44, antiCD49d and anti-CD29 (9EG7) that selectively recognizes activated b1 [42]. These antibodies did not block low HA binding of control LNC.…”

β1-Integrin Cytoplasmic Subdomains Involved in Dominant Negative Function

“…To investigate this possibility further, we used two antibodies, mAb 12G10 and mAb 9EG7, which recognize epitopes expressed only in the ligand-competent and ligand-occupied ␤1 (Lenter et al, 1993;Mould et al, 1995). Expression of these epitopes reflects changes in integrin ectodomain conformation, which can also be induced by exposing ␤1 to Mn ϩϩ (Bazzoni et al, 1995;Mould et al, 1995).…”

“…The following antibodies were used: the rat anti-human ␤1 mAb 13 (Akiyama et al, 1989) was a gift from K. Yamada (National Institutes of Health, Bethesda, MD); the activating mouse anti-human ␤1 mAb TS2/16 (Hemler et al, 1984) was obtained from American Type Culture Collection (Rockville, MD); the mouse anti-human ␤1 mAb 12G10 was characterized previously (Mould et al, 1995); the rat anti-mouse ␤1 mAb 9EG7, with human cross-reactivity (Lenter et al, 1993), was a gift from D. Vestweber (ZMBE Technologiehof, Muenster, Germany); the blocking mouse anti-human ␤1 mAb AIIB2 (Werb et al, 1989) was a gift from C. Damsky (Department of Stomatology, University of California, San Francisco, CA); the inhibitory PB1 mAb against hamster ␣5␤1 heterodimer was obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA); the blocking anti-mouse ␣V H9.2B8 mAb (Moulder et al, 1991) was purchased from PharMingen (San Diego, CA); the rat anti-mouse ␣6 mAb GoH3 (Sonnenberg et al, 1988) was a gift from A. Sonnenberg (The Neederland Cancer Institute, Amsterdam, the Netherlands); the anti-talin mAb 8d4 was obtained S.F. Retta et al…”

“…The following antibodies were used: the rat anti-human ␤1 mAb 13 (Akiyama et al, 1989) was a gift from K. Yamada (National Institutes of Health, Bethesda, MD); the activating mouse anti-human ␤1 mAb TS2/16 (Hemler et al, 1984) was obtained from American Type Culture Collection (Rockville, MD); the mouse anti-human ␤1 mAb 12G10 was characterized previously (Mould et al, 1995); the rat anti-mouse ␤1 mAb 9EG7, with human cross-reactivity (Lenter et al, 1993), was a gift from D. Vestweber (ZMBE Technologiehof, Muenster, Germany); the blocking mouse anti-human ␤1 mAb AIIB2 (Werb et al, 1989) was a gift from C. Damsky (Department of Stomatology, University of California, San Francisco, CA); the inhibitory PB1 mAb against hamster ␣5␤1 heterodimer was obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA); the blocking anti-mouse ␣V H9.2B8 mAb (Moulder et al, 1991) was purchased from PharMingen (San Diego, CA); the rat anti-mouse ␣6 mAb GoH3 (Sonnenberg et al, 1988) was a gift from A. Sonnenberg (The Neederland Cancer Institute, Amsterdam, the Netherlands); the anti-talin mAb 8d4 was obtained S.F. Retta et al from Sigma (St. Louis, MO); the anti ␣-actinin mAb 1682 was from Chemicon (Temecula, CA); rabbit polyclonal antisera to human fibronectin and to ␣V, ␣3, and ␣5 integrin cytoplasmic domains were produced in our laboratory (Tarone et al, 1984, Defilippi et al, 1992; the FAK 4 polyclonal antibody and the mAb FAK9.2 for FAK immunoprecipitation and Western blotting, respectively, were prepared in our laboratory as described previously (Defilippi et al, 1995); a mouse anti-paxillin mAb and the anti-phosphotyrosine mAb PY20 were purchased from Transduction Laboratories (Nottingham, UK); fluorescein-labeled phalloidin, fluorescein-labeled goat anti-mouse IgG, rhodamine-labeled goat anti-mouse IgG, and rhodamine-labeled goat anti-rabbit IgG were all from Sigma.…”