Eur. J. Immunol. 1987.17: 921-928 Monoclonal antibodies to rat B lineage-associated antigen 921 zy Frans G. M. Kroese, Auk S. Wubbena, Davine Opstelten, Gerrit Jan Deenen, Edwin H. Schwander', Loe De Leij', Hans VOS", Sibrand Poppema", Johanna Volberda and Paul Nieuwenhuis Departments of Histology, Clinical Immunology+ and Pathology++, University of Groningen, Groningen 1 Introduction Mature B lymphocytes constitute B lymphocyte differentiationin the rat: production and characterization of monoclonal antibodies to B lineage- associated antigens Three mouse monoclonal antibodies (mAb) directed against rat B lineage antigens were produced. The mAb, designated HIS14 (IgGI), HIS22 (IgM) and HIS24 (IgGZb), were characterized for binding to lymphoid and nonlymphoid tissues by immunoperoxidase staining of frozen sections and by (double-) immunofluorescence staining of single cell suspensions from lymphoid organs. HIS14 recognized a pan B cell determinant: it reacted with virtually all cells of each anatomic B cell compart- ment and with about 95% of surface (s)Ig+ cells in thoracic duct lymph and in suspensions of spleen and lymph nodes. HIS22 and HIS24 detected B lineage-associ- ated antigens expressed by major subpopulations of B cells. HIS22 predominantly stained the lymphocyte corona, but not (or weakly) the germinal centers and splenic marginal zones, whereas HIS24 reacted with both corona and germinal center and not (or weakly) with marginal zone. In accordance with this, substantial proportions of sIg' cells in spleen cell suspensions did not express HIS22 or HIS24 determinants (20% and 27%, respectively). In bone marrow the vast majority of cytomplasmic zy p ' pre-B cells were HIS14' and HIS24+, and up to one third also HIS22+, indicating an appearance of the determinants early in B lymphocytopoiesis. The antigens recog- nized by HIS14, HIS22 and HIS24 are lost during the final stage of B cell differentia- tion: none of the mAb bound to plasma cells. As far as detectable, neither cells of myeloid and erythroid lineages in bone marrow nor thymocytes were stained by HIS14, HIS22 or HIS24. In suspensions of peripheral lymphoid organs (spleen and lymph nodes) but not in thoracic duct lymph, HIS14 and HIS24 labeled a small proportion (12% and 14%, respectively) of Ig- cells. HIS22 did not bind to Ig- peripheral lymphocytes. Reactivity of HIS14, HIS22 and HIS24 with nonlymphoid tissues was virtually absent; HIS22 stained the high endothelial venules in lymph nodes and Peyer's patches. As determined by immunoblotting, the antigenic determinants on lymph node cells recognized by HIS14, HIS22 and HIS24 were present on molecules with an apparent molecular mass of 205 kDa, 210 (and 175) kDa and 205 kDa, respectively, which is similar to the molecular mass of the B cell form of the rat leukocyte common antigen. In addition, the antigens recognized by HIS14, HIS22 and HIS24 co-capped with the leukocyte common antigen. This suggests that each of the three mAb recognize determinants present on the B cell form of the leukocyte common antigen. cyte population means in terms of the physiology of the immune response. a population of cells heterogeneous in both function and phenotype [l, 21. In peripheral lymphoid organs heterogeneity is also evident from the existence of morphologically defined B lymphocyte com- partments (primary follicles, secondary follicles [lymphocyte corona and germinal center] and splenic marginal zone) [3]. While all B cells presumably have the capacity to respond to (antigenic) stimuli by proliferation and differentiation into plasma cells secreting immunoglobulins, it is, however, poorly understood what the observed heterogeneity of the B lympho- [I 59861 Correspondence: Frans G. M. Kroese, Department of Histology, Uni- versity of Groningen, Oostersingel 69/1, 9713 EZ Groningen, The Netherlands Abbreviations: BM: Bone marrow FITC: Fluorescein isothiocy- anate HEV: High endothelial venules mAb: Monoclonal antibody- (ies) LCA: Leukocyte common antigen(s) LN: Lymph node PBS: Phosphate-buffered saline PP: Peyer's patches sIg: Surface immu- noglobulins TDL: Thoracic duct lymphocytes TRITC: Tetrarnethyl rhodamine isothiocyanate In adult mammals, B lymphocytes are produced primarily in the bone marrow (BM) [4], deriving from pre-B cells contain- ing p heavy chains only in their cytoplasm [5-71. Little is known about the differentiation steps before the pre-B cell stage, partly due to the absence of well-defined surface mar- kers on the early (Ig-) B lineage-committed cells. For the study of both early and late B cell differentiation phenomena, the rat has shown to be an appropriate experi- mental animal [8, 91, in particular because two Ig zyx x light chain allotypes are known to exist [lo-121 (x chains account for more than 95% of the rat Ig light chains [13, 141). Congenic rats differing in their x allotype have been constructed [8, 151 and monoclonal antibodies (mAb) specific for these allotypes have been produced [8, 15-17]. To fully employ the rat as an experimental model, monoclonal antibodies (mAb) to B lineage-associated determinants would be of great help. Although many mAb for human [18-261 and some for mouse [27-301 B lineage-associated determinants have been described, few are available that react with B cell-associated determinants of the rat [31]. zyxw 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1987 0014-2980/87/0707-0921$02.50/0