B lymphocyte differentiation in the rat: production and characterization of monoclonal antibodies to B lineage‐associated antigens

Abstract

Three mouse monoclonal antibodies (mAb) directed against rat B lineage antigens were produced. The mAb, designated HIS14 (IgG1), HIS22 (IgM) and HIS24 (IgG2b), 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 compartment 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-associated 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 mu+ 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 recognized by HIS14, HIS22 and HIS24 are lost during the final stage of B cell differentiation: 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.

Biochemical and antigenic characterization of CD45 polypeptides expressed on plasma membrane and internal granules of human neutrophils

“…Interestingly, the expression of the different CD45 proteins on resident or circulating leukocyte cell subsets appears to be related with the cellular maturation stage [13,22]. However, the physiological significance of this selective expression still remains unknown.…”

Slow recovery of follicular B cells and marginal zone B cells after chemotherapy: implications for humoral immunity

“…For immunohistochemistry the following primary antibodies were used: ED1, ED2, ED3, ED5 (Serotec Ltd, Oxford, UK). T cell receptor (TCR) (Pharmingen, San Diego, CA, USA) and CD45R [22,23], CD90, IgM, IgD, HIS57 [21] and CD24 [24] ( Table 1). Secondary antibodies rabbit-antimouse immunoglobulins conjugated to peroxidase and goat-antirabbit immunoglobulins conjugated to peroxidase were obtained from Dako (Dako, Glostrup, Denmark).…”

Splenectomy of rats selectively reduces lymphocyte function-associated antigen 1 and intercellular adhesion molecule 1 expression on B-cell subsets in blood and lymph nodes

“…28 Both on cytospots and on cryostat sections 2 antigens were simultaneously revealed to identify proliferating B cells as described. 29 B cells were identified using the appropriate antibodies (His14, 25 IgM, and IgD 20 ) and visualized in blue. 30 After denaturation the BrdU ϩ cells were identified and visualized in red as described.…”

“…The first incubation step was performed using a monoclonal mouse antibody for B lymphocytes (His14). 25 For the second incubation step a cocktail was used, which consisted of goat antimouse IgG (Dianova, Hamburg, Germany) and rabbit anti-FITC. Finally a cocktail of alkaline phosphatase-antialkaline phosphatase (APAAP) mouse monoclonal and swine antirabbit was added to the sections.…”