Journal of Immunological Methods, 33 (1980) 11--21 11 © Elsevier/North-Holland Biomedical Press ROSETTE FORMATION WITH PROTEIN A-COATED ERYTHROCYTES: A METHOD FOR DETECTING BOTH IgG-BEARING CELLS AND ANOTHER SUBSET OF HUMAN PERIPHERAL BLOOD B LYMPHOCYTES S. ROMAGNANI, F. ALMERIGOGNA, GRAZIA M. GIUDIZI and M. RICCI Clinical Immunology Laboratory, University of Florence, Policlinico di Careggi, Viale Morgagni, 50134 Florence, Italy (Received 31 July 1979, accepted 23 October 1979) Human peripheral blood lymphocytes were tested for ability to form rosettes with human red blood cells (HRBC) coated with staphylococcal protein A (SpA-HRBC). Puri- fied T lymphocytes did not form rosettes, but in T-cell-depleted suspensions the number of cells forming rosettes with SpA-HRBC was significantly greater than in unfractionated suspensions. When non-T-cell suspensions were depleted of Ig-bearing lymphocytes, cells forming rosettes with SpA-HRBC were no longer detectable. The number of cells forming rosettes with SpA-HRBC in either unfractionated or T-cell-depleted suspensions was significantly higher than the number forming rosettes with HRBC coated with anti-human 7 chain immunosorbent-purified rabbit antibodies. Membrane components reacting with SpA-HRBC were not passively adsorbed on the cell surface and could be actively synthesized by lymphocytes. The SpA-reacting mem- brane components present on some B lymphocytes were sensitive to treatment with low concentrations of pronase, but other B cells maintained ability to form rosettes even after treatment with higher pronase concentrations. Incubation of T-cell-depleted lymphocyte suspensions with anti-human 3', and al~o with anti-human p or anti-human 5 chain F(abt)2 fragments induced significant reduction in the number of cells forming rosettes. After incubation of the same cell suspensions with a mixture of anti-')', anti-p and anti-5 (F(ab~)2 fragments, virtually all the lympho- cytes lost the ability to form rosettes with SpA-HRBC. These findings suggest that rosette formation with SpA-HRBC may be used as a method for detecting IgG-bearing cells but also to detect a subset of IgM- and/or IgD- bearing human B lymphocytes. INTRODUCTION It is well established that human and other mammalian IgG immuno- globulins are bound by the Cowan I strain of Staphylococcus aureus. The binding is due to the interaction of staphylococcal protein A (SPA) on the bacterial surface with the Fc region of the immunoglobulin molecule (Fors- gren and SjSquist, 1966; Kronvall and Williams, 1969). This reaction was found to be suitable for detection and quantitation of human lymphoid cells carrying IgG, either at the cell surface as receptors or attached to the mem-