Bone Marrow Subpopulations Research Articles

Identification of several cell surface proteins of non-T, non-B acute lymphoblastic leukemia by using monoclonal antibodies.

Monoclonal antibodies (MAb) were produced by immunization of BALB/c mice with cells from a non-T, non-B acute lymphoblastic leukemia (ALL) cell line. Nine distinct antigens (groups I to IX) were defined by these monoclonal antibodies, some of which appear to be associated with specific stages of cellular differentiation. The number of molecules of each MAb reactive with the ALL cell line, measured in a quantitative cellular radioimmunoassay, varied from 0.6 X 10(5) to 11 X 10(5) molecules/cell, indicating that the antigens identified represent major constituents of the cell surface. The biochemical nature of the antigens was examined on the ALL cell line by antibody affinity chromatography and/or immunoprecipitation and SDS-polyacrylamide gel electrophoresis. Groups I through III are composed of previously described antigens: HLA class I, HLA class II molecules, and CALLA, the common ALL antigen. The other MAb define antigens previously undescribed on non-T, non-B ALL cells. Group IV antigen is a polypeptide of apparent m.w. 95,000 distinct from CALLA. It is expressed on some ALL samples and on the vascular endothelial cells of several tissues. Group V antigen is a single polypeptide chain of m.w. 94,000, also distinct from CALLA and expressed by lymphocytes, thymocytes, acute myelogenous leukemia (AML) cells, and ALL cells. Group VI is a molecular complex composed of two noncovalently associated polypeptides of apparent m.w. 125,000 and 87,000 and appears to be restricted to ALL, AML, macrophages, and hematopoietic precursor cells. Group VII is a glycoprotein of apparent m.w. 85,000, which, within the thymus, is primarily restricted to the medullary area. It is also present on AML, bone marrow cells, and mature T and B lymphocytes. Group VIII is a disulfide-linked complex of apparent m.w. greater than 120,000 under nonreducing conditions. It is resolved into three major polypeptides of apparent m.w. 57,000, 47,000, and 41,000 under reducing conditions. This complex is found in greatest amounts on the non-T, non-B ALL cell line but is also present on AML, ALL, and on subpopulations of normal bone marrow and tonsil cells. Group IX antigen is a single polypeptide chain of apparent m.w. 51,000 on the ALL cell line. This antigen is expressed strongly on ALL and AML samples and on normal bone marrow; much lower antigenic density is found on thymus and tonsil cells. The antigens described here with a series of MAb produced in a single fusion represent a unique array of cell surface molecules of non-T, non-B ALL cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Proliferation of Infected Lymphoid Precursors Before Moloney Murine Leukemia Virus-Induced T-Cell Lymphoma

NFS/N mice inoculated with Moloney murine leukemia virus (M-MuLV) developed T-cell lymphoma after a 10-week latent period. Expression of lymphoid differentiation antigens, appearance of M-MuLV-encoded cell surface antigens, and rates of cellular proliferation were measured in splenic and bone marrow subpopulations during this latent period. At 2 weeks of age, Thy-1-and surface immunoglobulin-negative null cells of spleen and bone marrow expressed M-MuLV antigens whereas T- and B-lymphocytes did not. During the 3d and 4th weeks, the number of splenic null cells increased to six times the number found in uninfected controls. These null cells included the precursors of lymphocytes and hematopoietic cells. For the remainder of the latent period, the percentage of null cells undergoing proliferation was three times greater in the infected mice, while the total number of null cells remained constant. This proliferation was not accompanied by terminal differentiation or emigration of mature cell types from the spleen. Proliferation was substantially delayed in CBA mice, which are resistant to lymphoma induction.

Multiple splenic lymphoid cell subpopulations regulate H-2 antigen expression on teratocarcinoma cells in vivo.

Undifferentiated murine 402AX teratocarcinoma cells do not express MHC antigens when passaged in vitro or in vivo in genetically susceptible host mice. When passaged in vivo in genetically resistant mice, however, the tumor cells become H-2b antigen positive regardless of the H-2 haplotype of the resistant host mouse. The present studies use monoclonal anti-H-2b antibodies to corroborate these earlier findings, which were performed with conventional antisera. Previous studies have established that host bone marrow plus lymphoid cells from resistant primed donors regulate tumor cell H-2b antigen expression. Using bone marrow and mature lymphoid cell reconstitution techniques, the present studies indicate that splenic Ig- cells from genetically resistant host mice are the most efficient lymphoid cell subpopulation in tumor cell H-2b antigen induction. Ig+ spleen cells also reconstitute the capacity to induce teratocarcinoma cell H-2 antigens but are less effective than Ig- spleen cells. Tumor cell H-2 antigen induction in C57BL/6 beige mice is impaired compared to C57BL/6 hosts, which suggests that host NK cells may also be involved in tumor cell H-2 antigen induction. Reconstitution of lethally irradiated resistant hosts for teratocarcinoma cell H-2 antigen expression requires bone marrow plus resistant primed lymphoid cell subpopulations; bone marrow alone is insufficient. These results indicate that multiple splenic lymphoid cell subpopulations requiring a radiosensitive host environment and/or factor for differentiation regulate teratocarcinoma 402AX H-2b antigen expression in vivo in genetically resistant mice.

Murine cell surface glycoproteins. Purification of the polymorphic Pgp-1 antigen and analysis of its expression on macrophages and other myeloid cells.

We previously reported the initial characterization of a polymorphic major cell surface glycoprotein of about 80,000 daltons from mouse embryo 3T3 cells. This glycoprotein has now been purified 1800-fold to apparent homogeneity by monoclonal antibody affinity chromatography. The purified molecule retained the total antigenic activity of the cell, as determined by antibody binding assays. The quantity of the glycoprotein, 0.06% of the total protein of the crude cell extract, confirmed its presence as a major constituent of the cell plasma membrane. The monoclonal antibody was also used to detect related antigens in cells and tissues of C57BL/6J mice. The antigen was present in high concentration in macrophages and subpopulations of bone marrow and blood polymorphonuclear cells. Much lower concentrations of antigen were detected in spleen cells, thymocytes, and extracts of solid tissues. The apparent Mr of the target antigen of myeloid cells was 92,000. This molecule was a major surface constituent of myeloid cells with 10(6) antibody binding sites per cell containing 10% of total 125I incorporated by the lactoperoxidase procedure. The macrophage glycoprotein labeled on the cell surface with 125I was highly sensitive to trypsin, yielding an antigenically active soluble glycopolypeptide of about 65,000 daltons, that contained all of the incorporated 125I. A similar 65,000-dalton glycopeptide was released from 3T3 cells by trypsin cleavage. These data indicate that a major cell surface constituent of mouse myeloid cells is a 92,000-dalton glycoprotein closely related to the 80,000-dalton glycoprotein of mouse embryo 3T3 cells.

Qualitative and quantitative differences in intracellular protein patterns of normal immature myeloid and non-lymphocytic leukemic bone marrow cells

Polyacrylamide gel analysis of de novo synthesized polypeptides from non-lymphocytic leukemic blasts and normal immature myeloid bone marrow cells with comparable proliferating activity showed quantitative and qualitative differences, particularly in the high molecular weight region. Leukemic blast cells synthesized polypeptides between 94,000 and 200,000 Mr, not demonstrable in normal immature myeloid cells. Pulse-chase experiments revealed no further processing of these high molecular weight polypeptides nor excretion of such polypeptides out of the cells. Comparable high molecular weight polypeptides were present however in those normal bone marrow subpopulations which contain mainly mature myeloid cells. The presence of these high molecular weight polypeptides in leukemic cells, may reflect the activation of genome regions quiescent in normal immature cells and may represent a set of proteins comparable to those present in the maturing myeloid series of the normal bone marrow.

Osteogenesis and leukopoiesis within diffusion-chamber implants of isolated bone marrow subpopulations.

The developmental potential of isolated rabbit bone marrow cell populations was examined following autogeneous implantation into diffusion chambers. After 4 weeks, the implants were harvested and processed for light and electron microscopy. More total tissue was formed in abdominally implanted chambers than in corresponding femoral chambers. Two of the separated marrow cell populations produced significantly greater amounts of fibrous connective tissue, cartilage, and bone than did whole-marrow controls. These two populations, which were defined by density gradients of 1.050-1.055 gm/cm3 and 1.064-1.067 gm/cm3, consistently produced a fibrous connective tissue nodule in which were found dispersed foci of hyaline cartilage and woven bone. The denser population was distinguished further by the presence of leukopoietic foci in several of the implant chambers. Cartilage foci were found predominantly towards the center of the tissue nodules, whereas bone was dominant towards the periphery. Blood vessels, osteoclasts, bone remodeling, and mature lamellar bone were found only in those chambers which had been penetrated by the host's vascular system. The results indicate that 1) normal marrow tissue contains two separate osteoprogenitor cell populations; 2) these two progenitor populations represent separate osteogenic and chondrogenic capabilities; 3) one of these populations possesses a leukopoietic as well as an osteogenic potential; and 4) a competent vascular system is essential for the remodelling of bone into mature bone organs.

Subpopulations of Normal and Leukemic Human Thymocytes: An Analysis With the Use of Monoclonal Antibodies<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

Combinations of antibodies to membrane antigens and to terminal deoxynucleotidyl transferase (TdT) were used to study human thymocyte and bone marrow subpopulations and leukemia cells. Cortical thymocytes were TdT+ and expressed T-cell antigens (HuTLA+), a thymocyte-specific antigen (HTA-1+), and a leukocyte antigen (HLe-l++) but lacked detectable HLA-A,B,C and la (HLA-D) antigens. In contrast, medullary thymocytes were TdT-, HuTLA+, HTA-1-, HLe-l++. A small subpopulation of larger, probably immature, thymocytes were strongly TdT+, HuTLA+, la-, HTA-1-, HLe-l +/-. Many blast cells from cases of thymic acute lymphoblastic leukemia (Thy-ALL) showed the phenotype of this small subset, and only a proportion of Thy-ALL blast cells exhibited HTA-1 and HLe-l antigens as strongly as was observed on normal cortical thymocytes. In contrast, TdT+ cells observed in normal juvenile bone marrow were HuTLA, HTA-1-, HLA+, la+. This phenotype corresponded to the phenotype of the common form of ALL (non-T, non-B) and indicated that further studies are necessary to analyze the differentiation of bone marrow precursors to thymic cells.

Age-dependent alterations of DNA synthesis. Terminal deoxynucleotidyl transferase and DNA polymerase activities in bone marrow subpopulations from mice

The decrease of functional capacity of cellular immunity during ageing seems to be due to cellular changes of stem cells, particularly in the growth properties and the cell density in T-cell subsets. We approached this problem at the molecular biological level by quantifying the key enzymes necessary for DNA synthesis in bone marrow cells from mice: deoxynucleotidyl transferase (TdT) and DNA polymerase α. The bone marrow cells were fractionated on a discontinuous bovine serum albumin density gradient and the extractable enzyme activities (expressed per 10 8 nucleated cells in the respective fraction) were determined. TdT activity was found to decrease markedly during ageing. Mature animals contain only 34% and senescent animals only 13% of the activity observed in immature mice. From the density distribution analysis it was found that a shift of TdT-containing cells to the lower density occurs. The specific DNA polymerase α activity also decreases in bone marrow cells with age. While the overall activity amounts in immature cells to 78 enzyme units/10 8 cells, it decreases in mature cells to 57 units/10 8 cells, and in cells from senescent animals to 36 units/10 8 cells. Density distribution analysis of the cells shows that the highest activity is observed in the low-density fraction. From these experimental data we conclude that in the fractions containing precursor T-cells, a reduced number of proliferating cells is present.

Antigenic correlation between rat brain synaptic vesicles and rat bone marrow B lymphocytes.

Rabbit anti-rat brain synaptic vesicle serum reacted with thymocytes and B lymphocytes in cytotoxicity and immunofluorescence assays. Quantitative absorption analysis revealed that this antiserum contained antibodies specific for antigenic determinants on the surface membrane of a subpopulation of rat bone marrow B lymphocytes.

In vitro sensitization to transplantation antigens : IV. Density gradient centrifugation of sensitized cells and sensitization of density gradient-fractionated cells

C57 lymphoid cells were separated, by density centrifugation, into sub-populations of cells. The ability of these subpopulations to become sensitized in vitro to C3H alloantigens was studied. Of the five subpopulations of C57 lymph node, obtained after density gradient centrifugation, only the top, light density (A) and the middle, medium density (C) fractions are needed for in vitro sensitization. Upon in vitro sensitization, a change in density occurs and all the “active” cells appear in the top two (A and B) fractions of the gradient. Of the five subpopulations of C57 bone marrow, obtained after density gradient centrifugation, the top 2 (A and B) fractions are required for in vitro sensitization. Upon in vitro sensitization, a change in density occurs again but now the active cells are distributed among the intermediate bands, B and C. The results indicate that subpopulations of lymph node cells and bone marrow cells interact during in vitro sensitization to transplantation antigens.