Lymphoid Lines Research Articles

Differentiation analysis of bovine T-lymphosarcoma

The tumor cells from three different types (calf, skin and thymic) of bovine lymphosarcoma identified as sporadic bovine leukosis (SBL) were transplanted to nude mice and established as lymphoid tumor lines. To clarify the T-cell lineage, clonality and differentiation stage, these cell lines were examined for the immunophenotypic profile, the expression and the gene rearrangement of T-cell receptor (TCR) genes. The tumor cells from all three types showed α-naphthyl acetate esterase-positive staining and the absence of surface immunoglobulin (sIg). Moreover, the tumor cells appeared to be all BoCD5 + BoCD6 + BoCD4 − BoCD8 − cells in the FACS analysis with mAbs. Rearrangement of TCR-β and -δ, but not of TCR-α and -γ, genes was found to be unique for each tumor line showing T-cell clonality. Mature or truncated TCR-β, -γ and -δ transcripts were detected in the tumor cells, but all of them were found to be non-functional in cDNA cloning analysis. These results suggested that tumor cells from SBL are immature double-negative T-cells expressing non-functional TCR γδ transcripts to somewhat differentiated degrees.

Radiation response of mouse lymphoid and myeloid cell lines. Part II. Apoptotic death is shown by all lines examined.

The mode of death induced by gamma-irradiation in a panel of 10 mouse lymphoid or myeloid cell lines was examined. Four of these lines were known to lose viability (membrane integrity) rapidly after irradiation, whilst the others were known to lose viability considerably more slowly. However, based on the criteria of morphology and DNA degradation pattern, all 10 lines showed apoptotic death. The occurrence of apoptosis after irradiation in rapid-dying lymphoid cell lines was consistent with published results, whilst the demonstration of apoptosis in slow-dying lines was unexpected. Cells of the slow-dying lymphoid lines underwent one or more mitoses prior to death, a feature also reported for fibroblastoid cell lines. However, the occurrence of radiation-induced necrosis in fibroblasts suggests that the pathways leading to 'mitotic death' differ between fibroblastoid and lymphoid cell lines.

Reduced expression of major histocompatibility complex class I free heavy chains and enhanced sensitivity to natural killer cells after incubation of human lymphoid lines with β2‐microglobulin

Enhancement of major histocompatibility complex (MHC) class I expression leads to protection from recognition by natural killer (NK) cells in several systems. MHC class I gene products can be expressed in different forms at the cell surface--for example as "empty" beta 2-microglobulin (beta 2m)-associated heterodimers or free heavy chains. To study the role of different class I heavy chain forms in NK target interactions, we have used lymphoblastoid target cell lines preincubated with beta 2m. This was found to shift the equilibrium between beta 2m-associated and non-associated--heavy chains in favor of the former. In parallel, there was a significant increase in NK sensitivity. The recognition of MHC class I-deficient cell lines was not affected by beta 2m, arguing against a general nonspecific effect of beta 2m on NK sensitivity. Our data indicate that protection against NK recognition correlates with target cell expression of free heavy chains (i.e. devoid of beta 2m) rather than with expression of complexes.

Prolactin receptors are found on heterogeneous subpopulations of rat splenocytes

An increasingly large body of evidence implicates PRL as an immunoregulatory molecule. While most of the data relate to PRL levels and immunocompetence in vivo, we have shown that PRL is mitogenic for splenocytes from ovariectomized rats and rats in certain other hormonal states. This finding suggests that these lymphocytes express PRL receptors. Here, we wished to determine whether all or only a subset of splenocytes were PRL receptor positive. By using polyclonal as well as monoclonal antibodies to PRL receptor, we determined that as many as 20% of the primary splenocytes expressed PRL receptors. In a culture of Nb2 cells, a PRL receptor-positive lymphoid line, as many as 70% were PRL receptor positive. Dual labeling for lymphoid-specific antigen surface markers and PRL receptor indicated that about one third of the PRL receptor-positive splenocytes were kappa-light chain-positive B-cells, while the others stained with antibodies to T-cell markers, CD4 or CD8. These data confirm that lymphocytes express PRL receptors and show for the first time that PRL receptor-positive lymphocytes are a heterogenous subset of total primary splenocytes. These cells may be the target for PRL-mediated immunoregulation.

Prolactin receptors are found on heterogeneous subpopulations of rat splenocytes.

An increasingly large body of evidence implicates PRL as an immunoregulatory molecule. While most of the data relate to PRL levels and immunocompetence in vivo, we have shown that PRL is mitogenic for splenocytes from ovariectomized rats and rats in certain other hormonal states. This finding suggests that these lymphocytes express PRL receptors. Here, we wished to determine whether all or only a subset of splenocytes were PRL receptor positive. By using polyclonal as well as monoclonal antibodies to PRL receptor, we determined that as many as 20% of the primary splenocytes expressed PRL receptors. In a culture of Nb2 cells, a PRL receptor-positive lymphoid line, as many as 70% were PRL receptor positive. Dual labeling for lymphoid-specific antigen surface markers and PRL receptor indicated that about one third of the PRL receptor-positive splenocytes were kappa-light chain-positive B-cells, while the others stained with antibodies to T-cell markers, CD4 or CD8. These data confirm that lymphocytes express PRL receptors and show for the first time that PRL receptor-positive lymphocytes are a heterogenous subset of total primary splenocytes. These cells may be the target for PRL-mediated immunoregulation.

Cell cycle effects of the DNA topoisomerase inhibitors camptothecin and m-AMSA in lymphoblastoid cell lines from patients with Fanconi anemia

Patients with the autosomal recessive disorder Fanconi anemia (FA) present with progressive pancytopenia, skeletal abnormalities and a predisposition to leukemia. In addition to elevated rates of spontaneous chromosome aberrations occurring in cultured fibroblasts and lymphoblastoid cell lines, an increased susceptibility to DNA cross-linking agents and oxygen has been found. To explain this hypersensitivity to clastogenic agents a defective function of DNA topoisomerase I or II could be invoked, a suggestion which is supported by the co-localization of the DNA topoisomerase I gene and a putative FA gene to chromosome 20q. In order to investigate the function of DNA topoisomerases in FA, the sensitivity of lymphoid B-cell lines derived from FA patients and control cell lines to inhibitors of DNA topoisomerases I and II was compared using continuous bromodeoxyuridine labeling and bivariate Hoechst/ethidium bromide flow cytometry. Both agents inhibited cell proliferation mainly by arresting cells in the G2 phase of the cell cycle. However, no difference was found in sensitivity towards both DNA topoisomerase inhibitors between control and FA cell lines.

Specific protein dephosphorylation in apoptosis induced by ionizing radiation and heat shock in human lymphoid tumor lines.

Apoptosis or programmed cell death is observed in a variety of organisms and tissues and is characterized by distinct morphologic changes to the cell. Although early indicators of this process have been described, their functional relevance remains unknown. We have used two-dimensional gel electrophoresis to look for characteristic and consistent changes in the phosphorylation state of proteins during apoptosis, induced by different agents, in the B cell lymphoma line, BM13674, and the T cell leukemia line, CEM-C7. We report that apoptosis induced by either heat treatment or by ionizing radiation exposure is accompanied by dephosphorylation of a limited number of specific proteins. The pattern of dephosphorylation was similar after both treatments in BM13674 cells. In CEM-C7 cells, dephosphorylation was also observed after heat and irradiation. One of these proteins corresponded to one dephosphorylated in BM13674 cells. Okadaic acid, an inhibitor of phosphatases 1, 2A, and, to a lesser extent, 2B, prevented apoptosis in all cases and inhibited dephosphorylation of this common protein as well as some of the others. It seems likely that activation of a phosphatase(s) or loss of activity of a kinase is of central importance in apoptosis in these systems.

Isolation of the JMH Antigen on a Novel Phosphatidylinositol-Linked Human Membrane Protein

AbstractJMH is a high-frequency human erythrocyte blood group antigen. Previous work has shown that JMH is absent from complement-sensitive erythrocytes of patients with paroxysmal nocturnal hemoglobinuria (PNH); such cells have a broad defect in expression of phosphatidylinositol (PI)-linked proteins. Using both human JMH antisera and a JMH-like murine monoclonal antibody, we have identified a 76-Kd membrane protein present in JMH-positive but not JMH-negative erythrocytes. A similar 76-Kd JMH protein was also identified on a human lymphoid T-cell line, HSB-2. Using PI-specific phospho-lipase C, a small amount of JMH antigen could be cleaved from intact erythrocytes and immunoprecipitated from the supernate of treated erythrocytes, thus confirming that the protein bearing the JMH antigen is anchored by a PI-linkage to the erythrocyte membrane. This protein was further shown not to be identical to decay accelerating factor (70 Kd), a previously identified PI-anchored protein of somewhat similar molecular weight.

Isolation of the JMH antigen on a novel phosphatidylinositol-linked human membrane protein

JMH is a high-frequency human erythrocyte blood group antigen. Previous work has shown that JMH is absent from complement-sensitive erythrocytes of patients with paroxysmal nocturnal hemoglobinuria (PNH); such cells have a broad defect in expression of phosphatidylinositol (PI)-linked proteins. Using both human JMH antisera and a JMH-like murine monoclonal antibody, we have identified a 76-Kd membrane protein present in JMH-positive but not JMH-negative erythrocytes. A similar 76-Kd JMH protein was also identified on a human lymphoid T-cell line, HSB-2. Using PI-specific phospholipase C, a small amount of JMH antigen could be cleaved from intact erythrocytes and immunoprecipitated from the supernate of treated erythrocytes, thus confirming that the protein bearing the JMH antigen is anchored by a PI- linkage to the erythrocyte membrane. This protein was further shown not to be identical to decay accelerating factor (70 Kd), a previously identified PI-anchored protein of somewhat similar molecular weight.

Binding receptors for alpha-L-fucosidase in human B-lymphoid cell lines.

An established mechanism for directing newly made acid hydrolases to lysosomes involves acquisition of mannose 6-phosphate residues by the carbohydrate portion of acid hydrolases followed by binding to specific membrane-bound transport receptors and delivery to lysosomes. Two distinct phosphomannosyl receptors (CI-MPR and CD-MPR) have been identified. Alternative mechanisms for trafficking acid hydrolases exist. This report examines means for the possible receptor-mediated intracellular transport of alpha-L- fucosidase in lymphoid cells. The binding of alpha-L-fucosidase to intact cells and to total cell membrane preparations, in conjunction with immunoassays of solubilized membrane preparations, revealed the presence of CI-MPR and CD-MPR on human lymphoid and fibroblast cell lines. The mean level of CD-MPR in nine lymphoid cell lines was 7.2-fold greater than CI-MPR. The mean level of CI-MPR in two fibroblast lines was 3.8-fold greater than CD-MPR. The mean content of CI-MPR was 19.5-fold greater in the fibroblasts than in the lymphoid cells. The CD-MPR content of fibroblasts and lymphoid cells was nearly equivalent. Among these cell lines were a fibroblast and a lymphoid line from the same individual. These results indicate that human B-lymphoid cells are deficient in CI-MPR and suggest that modulation of expression of CI-MPR and CD-MPR in lymphoid cells differs from that in fibroblasts, including cell lines with identical genomes. No specific receptor capable of binding alpha-L-fucosidase independent of mannose 6-phosphate was demonstrable, despite published results that support the existence of a mannose 6-phosphate independent trafficking mechanism in lymphoid cells for this enzyme.

Identification of a 107-kD glycoprotein that mediates adhesion between stromal cells and hematolymphoid cells.

The mechanism of cell complex formation between lymphocytes and stromal cells was investigated. We found that lymphoid lines of both T and B lineages could form cell complexes with stromal cells from the thymus as well as bone marrow but not with macrophages or typical fibroblast lines. Formation of these cell complexes is temperature dependent and requires the presence of Mg2+, active cellular metabolism, and microfilament assembly of cytoskeleton. We raised an antiserum against a thymic stromal cell clone (BATE-2) in rats and found that, after absorption, this serum could effectively block cell complex formation between lymphocytes and stromal cells from both thymus and bone marrow. An efficient blocking was obtained only when the antiserum was added at the initial stage of cell interaction. From the blocking experiments and the SDS-PAGE analysis of immunoprecipitated materials from the stromal cell surface, we identified a unique 107-kD glycoprotein on the stromal cells as a molecule for mediating stromal cell-lymphocyte interaction. This is further supported by the findings that an antiserum raised in hamsters against the excised gel band corresponding to 107 kD, which specifically immunoprecipitated the 107-kD molecule, effectively blocked the lymphocyte-stromal cell interaction. The possible function of this molecule in hematolymphoid development is discussed.

Dexamethasone‐induced killing of neoplastic cells of lymphoid derivation: Lack of early calcium involvement

The role of calcium influx in dexamethasone-induced fragmentation of DNA was studied in the glucocorticoid-sensitive human lymphoid line of T cell derivation (CEM-C7). Reduction of calcium content in the medium or the use of EGTA increased DNA fragmentation and appeared to slightly enhance the effect of dexamethasone. Incubation of isolated nuclei in the presence of high concentrations of calcium did not bring about significant DNA fragmentation. Calmidazolium, an antagonist of calmodulin dependent reactions did not reduce the sensitivity of CEM-C7 cells to dexamethasone nor did it modify the response to dexamethasone of the resistant CEM-C1 line. It appears that in contrast to rodent thymocytes, massive calcium influx is not per se responsible for the initiation of directed cell killing (apoptosis).

Cytogenetic characterization of 20 lymphoblastoid lines derived from human individuals differing in bleomycin sensitivity

Forty lymphoblastoid (lymphoid) lines were established from 42 volunteer blood donors, including healthy individuals and patients with head and neck carcinomas. Each peripheral blood sample was split into two portions, one for the establishment of a lymphoid line and the other for short-term culture, which was used to estimate bleomycin sensitivity by cytogenetic procedures. Twenty lymphoid lines were selected at random to compare bleomycin sensitivity with data obtained from short-term lymphocyte cultures. In each set, bleomycin sensitivity of lymphoid cells was similar to that of the lymphocytes. The lymphoid lines, which can be propagated for an unlimited supply of relatively homogeneous cellular material, will be useful for a variety of future investigations.

Conversion of Normal Ly‐1‐Positive B‐Lineage Cells into Ly‐1‐Positive Macrophages in Long‐Term Bone Marrow Cultures

We obtained eight different cell lines in the long-term bone marrow culture system that showed a germ-line configuration of the joining (J) region segments of the Ig heavy-chain (IgH) genes. Their surface markers were CD45R+, Ly-1+, Lyb-2+, cIgM-, sIgM-, Ia-, Thy-1-, Mac-1-, and IL-2R (Tac)+. Use of very young mice and the presence of IL-5 were important for preferential promotion of the survival of B-lineage lymphocytes bearing the Ly-1 markers. When we treated two of them (J8 and J10) with 5-azacytidine for 24 h followed by co-culture with stromal cells and IL-.5, they became Ly-1+, sIgM+ B cells, and Ly-1+, Mac-1+ macrophagelike cells, respectively. After other early lymphoid lines (J1, J8, and J13) were maintained by co-culture with ST2 and IL-5 for more than a year, they showed a heterogeneous DNA rearrangement profile of the J region segment of the IgH gene, although only J13 rearranged the κ-light chain gene. Northern blot analysis revealed that these cell lines expressed Cμ-mRNA, and λ5-mRNA, consistent with normal pre-B cells. Intriguingly, J1, J8, and J13 expressed c-fms mRNA constitutively. When J13 cells were co-cultured with ST2 and GM-CSF in place of ST2 and IL-5, they acquired Mac-1 expression and retained Ly-1 expression. They were morphologically macrophages, nonspecific-esterase-positive, and showed phagocytosis of latex beads. These results support evidence for a close relationship between the myeloid and Ly-1+ B-cell pathways of differentiation, and indicate that our IL- 5-dependent clones are multipotential intermediates in differentiation from pro-B cells to B cells and macrophages.

Structure and expression of the T cell receptor gamma locus in pre‐B and early hemopoietic cells

The genetic structure and expression of the T cell receptor (TcR) loci were examined in pre-B and early hemopoietic cells. Thirty-eight percent of Abelson murine leukemia virus-transformed pre-B cell lines were rearranged at TcR gamma. Moreover, many pre-B cell lines were rearranged at two distinct gamma loci, V gamma 1.2 and V gamma 2. The gamma rearrangements in the pre-B cell lines were similar to those observed previously in T cell lines. V + C-containing gamma mRNA was detected in two pre-B cell lines. In all other pre-B and interleukin (IL) 3-dependent lymphoid and myeloid lines examined, smaller C gamma-containing mRNA were detected. These C gamma transcripts were independent of the genetic configuration of the gamma locus. In contrast, the TcR alpha and beta loci were in the germ-line configuration in all non-T cell lines examined and mRNA encoding these loci were not detected. When IL3-dependent lymphoid and myeloid cell lines were transformed to growth factor independence by a non-autocrine mechanism, no mRNA transcripts encoding TcR C gamma were detected. However, TcR C gamma mRNA transcripts were detected in factor-independent cell lines that arose by an autocrine mechanism. The cell cycle expression of C gamma was compared with protooncogenes and other marker genes previously shown to be cell cycle specific. mRNA transcripts encoding C gamma were detected in the highest amounts 4-8 h after IL 3, but not phorbol myristate acetate, addition. A similar time period of expression was observed with ornithine decarboxylase which has been shown to be expressed in G1 phase. These observations indicate that TcR gamma is often rearranged in pre-B cell lines and may be directly regulated by IL3 in IL3-dependent cells.

A T lymphoid cell line responds to a thymic stromal cell line by expression of Thy-1 and CD4.

We have cloned both T lymphoid and stromal lines from a single murine thymic tumor that was induced by a retrovirus carrying the v-myc oncogene (M-MuLV(myc]. The T lymphoid line, L4, was cloned by growth in agar. L4 cells were initially negative for Thy-1.2 and CD4 (although they contained rearranged TCR-beta genes), and they remained so if passaged in medium alone. However, cocultivation of these Thy-1.2- CD4- cells with the cloned stromal cell line, St3, resulted in sequential expression of Thy-1.2 and CD4 in subpopulations of cells. Thy-1.2+ CD4- and Thy-1.2+ CD4+ L4 subclones were obtained from the cocultures by subsequent cloning in agar. Derivation of these subclones from the starting Thy-1.2- CD4- clone was verified by Southern blot analyses specific for TCR-beta gene rearrangements and for M-MuLV(myc) proviral integration sites. Continuous cocultivation of Thy-1.2+ CD4+ L4 subclones with the St3 stromal cells was necessary for maintenance of CD4 on the cell surface. Furthermore, CD4 expression which was lost when CD4+ L4 cells were removed from the stroma could be reinduced if they were again cultured on St3 stroma. These cells may provide a model system for studying thymocyte-stromal cell interactions in induction and maintenance of expression of Thy-1 and CD4 molecules.

Suppression of MHC class I gene expression by N-myc through enhancer inactivation.

Amplification of the N-myc oncogene in human neuroblastoma is associated with increased metastatic ability. We previously found that over-expression of N-myc in rat neuroblastoma tumor cells causes a dramatic reduction in the expression of MHC class I mRNA. We show here that two distinct elements in the promoter render the MHC class I genes susceptible to N-myc-mediated suppression, one of which was identified as the MHC class I gene enhancer. Our data indicate that elevated N-myc expression is associated with reduced binding of a transcription factor that activates this enhancer. As a result, the activity of the MHC class I gene enhancer is greatly diminished. Elevated expression of the N-myc oncogene in human neuroblastomas and murine pre-B lymphoid lines also correlated with reduced factor binding to the MHC class I gene enhancer. Thus, an important effect of N-myc may be to impair the function of certain cellular enhancers by altering the levels of their cognate binding proteins.

Propagation of reticuloendotheliosis virus strain t in cell lines derived from avian lymphoid leukosis lymphoma and Marek's disease tumour

Propagation of reticuloendotheliosis virus strain T (REV-T) was studied in avian lymphoid leukosis lymphoma-derived cell lines (LSCC-1104x5 and BK 3) and Marek's disease tumour-derived cell lines (MDCC-MSB1 and RP 1). These four cell lines showed cellular growth for a long time even after they were infected with REV-T. Production of non-oncogenic REV-T, replication non-defective virus, in these cell lines continued for 3 to 7 months. When culture fluids from 1104x5, MSB1 and RP 1, which were infected with oncogenic REV-T, were inoculated into 1-day-old chickens, acute development of lethal reticuloendotheliosis (RE) tumours occurred. Especially after the use of the fluids from 1104x5, propagation of oncogenic REV-T was observed for a long time, 7 months. Of the chickens inoculated with the culture fluid of any REV-T-infected cell lines, many chickens which survived for 4 weeks showed abnormal feathering, 'nakanuke'. The body weight of these chickens was significantly less than that of uninoculated chickens. No contamination with infectious REV was observed in the culture fluids from any uninfected line cells.

Adhesion of lymphoid cells to fibroblasts in tissue culture

In this study we have examined the cellular and molecular specificity of lymphocyte interaction with fibroblasts. Using mitogen-activated T-cells, we found that attachment to fibroblasts was highly sensitive to protease treatment, and to an antibody raised against the purified lymphocyte plasma membrane, but it was not mediated by the MEL-14 surface antigen or phosphomannosyl receptors. Lymphocyte interaction with fibroblasts was also unaffected by monoclonal antibodies against the LFA-1, Mac-1, and Class II MHC antigen complexes. In contrast, adhesion of both T- and B-lymphocytes was strongly inhibited by fucoidan, a polymer of sulphated fucose, whereas fucose, mannan, and mannose 6-phosphate had no effect. Both B- and T-lymphoid cell lines were able to recognise and adhere to fibroblasts, although the marked differences between the attachment of the different types of cell did not appear to be related to their immunological function. The attachment of most of the cell lines was prevented by the presence of fucoidan, whereas the inhibition of binding of each of the lymphoid lines in the presence of the anti-T-lymphocyte plasma membrane antibody varied widely. These findings suggest that lymphocyte attachment to fibroblasts involves multiple cell surface receptors, and that these are expressed at different levels on specific T- and B-cells.

Immunology and Cellular Biology of Hodgkin’s Disease

Patients with Hodgkin's disease, either untreated or in remission, exhibit a persistent defect in cellular immunity. This cellular immune defect appears to be the result of increased sensitivity to suppressor monocytes and T-suppressor cells, in addition to abnormal Interleukin-2 production. T-lymphocyte function is abnormal in patients with advanced disease. The precise origin of Reed-Sternberg and Hodgkin's cells is unknown. Reed-Sternberg cells function as antigen-presenting cells and as accessory cells in mitogen-induced T-cell proliferation. They have properties in common with dendritic cells and activated lymphocytes. L428 cells express a transformation-associated phosphorylated transmembrane protein, with properties of a growth factor receptor, that may play a role in tumorigenic transformation.