Eur. J. Immunol. 1989.19: 323-328 Common T and B cell activation antigen 323 z Angela Rho, Maria Elisabetta Cosulich, Anna Rubartem, Maria Rosaria Mazza and Antonio Bargellesi Istituto Scientific0Tumori and Istituto di Chimica Biologica, Universith di Genova, Genova MLR3 molecule is an activation antigen shared by human B, T lymphocytes and T cell precursors* MLR3 molecule is a membrane glycoprotein (mol. mass range 28-34 kDa) present on activated, but not resting human peripheral T cells, B cells and thymocytes. Its kinetics of appearance on the cell surface (3 h after the addition of the inductive signal to the cells) suggests that it is an early activation antigen. The proliferative response of cultured T and B lymphocytes and thymocytes to different activation signals is in- hibited by the addition of MLR3 monoclonal antibody. Moreover the antibody in combination with non-mitogenic doses of phorbol myristate acetate leads to prolifera- tion of thymocytes and resting B and T lymphocytes. In the latter, synthesis of interleukin 2 is also induced. Biochemical analysis of MLR3 antigen indicates that it is a phosphorylated protein with N-linked sugar moieties. Together these data suggest a role for MLR3 antigen in the signal transduction process during activation, both for mature lymphocytes and for T cell precursors. 1 Introduction In recent years the activation process of human lymphocytes has been studied in detail regarding membrane antigens and the biochemical events involved in the signal transduction mechanisms. Besides the antigen receptor, other activation pathways have been defined by means of monoclonal anti- bodies (mAb) against cell surface molecules: in T cells, in addition to the CD3-Ti structures [l-31, CD2 [4], CD28 zyxwvu [5,6], Tp 103 [7] and zyxwvutsr 5/9 [8] molecules mediate proliferation signals when they interact with the corresponding antibodies. Simi- larly, human B cells can be activated either by anti-p-chain antibodies [9], or by an alternative stimulus delivered by a mAb to CD20 antigen [lo-111. By contrast, purified human thymocytes fail to respond to most stimuli able to trigger mature T lymphocytes and only the CD2 molecule can repre- sent an activation pathway [12]. Recently, we have reported on an activation antigen called MLR3 present on activated but not resting T lymphocytes and thymocytes. The MLR3 molecule is likely involved in the early steps of T cell activation since the proliferative response of resting T cells to anti-CD3-Sepharoseand interleukin (IL 1) or accessory cells, but not the IL 2-dependent T cell prolifera- tion, is inhibited by MLR3 mAb [13]. In the present study we demonstrate that MLR3 molecule is expressed also by activated (but not resting) B lymphocytes and that the early proliferative response of B cells to various [I 69991 zyxwvu * zyxwvutsrqpo This work was supported by a grant from C.N.R. Progetto finaliz- zato Ingegneria Genetica e basi molecolari delle malattie ereditarie N. 87.00877.51 and by a grant from C.N.R. Progetto finalizzato Oncologia Anticorpi Monoclonali ed antigeni di membrana N.771 87.01174. Correspondence: Angela Risso, Istituto scientific0 Tumori. Wale Benedetto XV, 10, Genova, Italy Abbreviations: mAb: Monoclonal antibody(ies) PMA: Phorbol12- myristate 13-acetate Ig: Immunoglobulin IL 2: Interleukin 2 SAC: Staphylococcus aureus Cowan I PBL Peripheral blood mononuclear cells SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis stimuli is inhibited by the addition of MLR3 mAb to the cul- tures. Following MLR3 expression induced by non-mitogenic doses of phorbol myristate acetate (PMA), MLR3 mAb is able to trigger proliferation of B, T lymphocytes and thymocytes. Furthermore, both 28- and 34-kDa bands immunoprecipitated by MLR3 mAb are phosphorylated in activated cells. In human T cells MLR3 antigen is phosphorylated in response to different concentrations of PMA and to phytohemagglutinin (PHA). Thus, MLR3 is a molecule common to T, B lympho- cytes and thymocytes, expressed on the membrane surface in the early activation steps and involved in the signal transduc- tion mechanisms. 2 Materials and methods 2.1 Antibodies MLR3 mAb (a mouse IgGI) was purified from ascites by 40% saturated ammonium sulfate precipitation followed by gel fil- tration with AcA 34 Ultrogel (LKB, Bromma, Sweden) Anti- Leu-11 mAb was obtained from Becton-Dickinson (Italia Spa), OKT3 and OKMl mAb were purchased from Ortho Diagnostics (Raritan, NJ). Anti-CD3, anti-CD2 and anti-CD4 mAb-producing cell lines were obtained from the American Type Culture Collection (Rockville, MD). BT2/9 (an anti- HLA class I1 mAb) was a gift of Dr. G. Corte (Genova). Anti- CD3 of the IgM isotype was gently provided by Dr. E. Reinherz (Boston). An anti-human plasma fibronectin mAb (a mouse IgG1) was a gift of Dr. L. Zardi (Genova). A rabbit anti-human IgM (hIgM) was obtained by Cappel (Cochran- ville, PA). Purified immunoglobulin (Ig) was derived from ascites as described above. A fluorescein isothiocyanate-con- jugated goat anti-mouse Ig antiserum was prepared as described [13] and used as a second reagent in indirect immunofluorescence assays. 2.2 Cell preparation Peripheral blood mononuclear cells (PBL) were isolated from heparinized blood by standard Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) centrifugation. T cells were separated by rosetting as previously described [13]. Recovered cells were > 99% CD3+, as determined by immunofluorescence. B lym- phocytes were recovered from tonsil cell suspensions depleted Q VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1989 0014-298018910202-0323$02.50/0