Enrichment of human marrow lymphocytes with monoclonal antibodies to murine antigens.

Abstract

Two monoclonal antibodies, prepared against a murine B lymphoma and characterized as binding to a cell surface antigen represented primarily on cells ofthe B lineage, were found to bind to human hemopoietic cells. These antibodies recognize similar populations of cells in mice and humans. Antibodies from clones 177.17 and 83.4 bound to 6% of human bone marrow nucleated cells. This included all cells with detectable cell surface Ig (sIg') and those that lack sIg but have detectable cytoplasmic ,u (sIg, cIA+), considered to be the immediate precursors of B lymphocytes (pre-B cells). In addition, these antibodies bound to a subpopulation of T cells and a proportion of null lymphocytes in marrow, spleen, and peripheral blood. An unexpected finding was that established pre-B cell lines were not recognized by these antibodies and possible reasons for this are considered. By using antibody-coated polystyrene plates for cell depletion and recovery, highly enriched preparations of ciA+, sIg-cells have been obtained. These antibodies and enrichment procedures should prove valuable in establishing the minimal requirements for maturation of these putative precursors in vitro, for comparative studies of immunodeficiency/autoimmune diseases in man and experimental animal models, and for monitoring the outcome of therapeutic marrow transplantation.A panel ofmonoclonal rat antibodies was recently prepared and characterized as directed to determinants on murine cells of B cell lineage. One group of seven clones produced antibodies that did not recognize immunoglobulin and that similarly bound to a series of established murine cell lines. Five of these made antibodies ofsatisfactory avidity and were studied in detail. The antigen(s) recognized by these antibodies is displayed on most B cells, a subpopulation of peripheral T cells, and a proportion of antibody-secreting cells (1). In addition, cell surface immunoglobulin negative (sIg-) A number ofcell surface molecules that were originally identified on murine leukocytes have been found to be analogous to human antigens. Often the basic structures-and in some cases even the cellular distributions-of these have been comparable, and there are several recent examples in which heteroantibodies and alloantibodies were found to detect conserved antigen determinants (3-9).We report here that two of our monoclonal antibodies that were prepared against murine cells bind also to human leukocytes and do so with remarkably similar selectivity for lymphocyte sets. Furthermore, these reagents can be used in the same way as with murine cells to obtain highly enriched preparations of sIg-human marrow lymphocytes. MATERIALS AND METHODSCell Suspensions. Peripheral blood and bone marrow cells were obtained from healthy adult volunteers. Histologically normal spleen samples were obtained following therapeutic splenectomy. Peripheral blood mononuclear cells were prepared by using Ficoll/Paque according to manufacturer's directions (Pharmacia). Enriched granulocytes were recovered from the ...

Cycling of peripheral blood and marrow lymphocytes in cyclic neutropenia.

“…In grey collie dogs, the animal model of this disease, the term canine cyclic hemopoiesis (CCH) is used because the neutropenia is always associated with cyclic monocytosis, reticulocytosis, and thrombocytosis. The periodicity in CCH usually approximates [11][12][13] days (5).…”

“…During our study, the patient had peripheral blood counts and differential counts every other day (at the same hour) for 50 days and was examined every week. Detailed studies of the peripheral blood and marrow were done on days 13,20,27,35,42, and 49.…”

“…Bone marrow samples were aspirated from the posterior superior iliac crest into a heparinized syringe. Bone marrow cell suspensions were freed of erythrocytes by resuspending pelleted cells in 10 ml of Tris HClbuffered 0.15 M NH4Cl (pH 7.4) at 40C until satisfactory erythrocyte lysis was achieved as described (13). Loss of nucleated cells with this procedure was always insignificant.…”

“…Cells for visual cell surface immunofluorescence were incubated with appropriate monoclonal antibodies as described in detail (13). Binding of the mouse monoclonal antibodies was detected with a FITC-conjugated goat anti-mouse Fab preparation that gave no detectable background staining on any of the samples studied.…”

“…Bone marrow CFU-c were assayed as described (13) by using 10% GCT conditioned medium (GIBCO) as a source of colony-stimulating factors. Marrow cultures were established in soft agar at a concentration 2 X 105 cells per ml, incubated for 7 days at 370C in 7% CO2 in humidified air, and scored for the presence of colonies consisting of >40 cells and clusters of 3-40 cells under a phase-inverted tissue culture microscope.…”