Steinar Funderud

Characterization of Human Mononuclear Cells after Positive Selection with Immunomagnetic Particles

We have investigated the possibility to employing magnetic monodisperse polymer particles for positive selection of human peripheral blood mononuclear cell populations. By carefully titrating the ratio between particles and cells we succeeded in isolating a number of cell populations that could be cultivated subsequently in vitro for functional studies. The success of the procedure is partly dependent on the properties of the monoclonal antibodies used to sensitize the cells. Provided these antibodies do not read with membrane structures involved in the transduction of activating signals, highly purified, quiescent cell populations can be recovered in a single fractionation step. In most instances panicles will detach from the isolated cells by overnight culture, and the particles can then be removed from the system by a suitable magnet. T lymphocytes, subpopulations of I lymphocytes, and B lymphocytes have been isolated in this way and studied in a variety of functional assay systems. Comparison with cells obtained after negative selection clearly demonstates the usefulness of this technique, especially if the membrane marker selected for it is not directly engaged in the activation processes.

Transforming growth factor type β (TGF β) inhibits G1 to S transition, but not activation of human B lymphocytes☆

Abstract Type β transforming growth factor (TGF β) is a polypeptide that may influence the growth of a variety of cell types in a positive or negative fashion. In this study we show that TGF β markedly inhibits DNA synthesis in normal and neoplastic human B lymphocytes stimulated to proliferate with anti-immunoglobulins and B-cell growth factor (BCGF). Although TGF β was needed during the initial 12 h of the culture to promote optimal inhibition, we found that it had little or no effect on several early to intermediate parameters of cell activation ([Ca 2+ ] i increase, c- myc mRNA increase, cellular enlargement, RNA increase, and the increase in the expression of the 4F2 activation antigen). In contrast, TGF β almost completely blocked the induction of transferrin receptor expression, which normally occurs in the late G 1 phase of the cell cycle. Therefore, we conclude that TGF β treatment leads to arrest of the cells in the middle to late G 1 phase, prior to transferrin receptor expression.

A new method for detachment of Dynabeads from positively selected B lymphocytes

This paper describes a method for the detachment of immunomagnetic beads from positively selected human B lymphocytes. After rosetting of B cells using anti-CD19 coated magnetic beads (Dynabeads M-450 Pan B, Dynal), the Dynabeads were rapidly detached (efficiency 80%) from the cells using goat anti-mouse-Fab antiserum (DETACHaBEAD, Dynal) at ambient temperature. Isolated B cells did not show significant differences in the expression of a number of B cell antigens when compared to B cells stained in fresh whole blood. In contrast, positively selected B cells that had detached from the beads following overnight incubation, demonstrated a significantly reduced expression of certain of the antigens examined (CD19, CD20 and CD23). It was further demonstrated that neither anti-CD19 nor anti-Fab resided on the surface of the cells after detachment. The cells were still in G0 phase (greater than 90%) at the end of the isolation procedure. Moreover, anti-IgM antibodies stimulated the vast majority of the cells to leave the G0 phase, and to progress through S phase in the presence of growth factors. The cells could also be stimulated to differentiate, further confirming the normal functional capacity of the isolated cells. The method described in this paper can also be used for the detachment of other positively selected cells, such as CD4+ T cells, CD8+ T cells and CD34+ stem cells.

Characterization of two murine monoclonal antibodies reactive with human B cells. Their use in a high-yield, high-purity method for isolation of B cells and utilization of such cells in an assay for B-cell stimulating factor

We describe two monoclonal antibodies, HH1 and HH2. Both reacted selectively with surface immunoglobulin (slg)‐positive human B cells. Both antibodies stained on average 7–8% of peripheral blood mono nuclear cells. They have not been found to react with cells or cell lines of other haematopoietic cell lineages, except that HH2 was positive on a small percentage of cells of the erythroid cell line K562. The molecular weight of the HH1 antigen was 95 kD, as established by Western blotting. Neither of these two antibodies reacted with Ig determinants, Fc receptors, complement receptors, or known class‐I or class‐II molecules. A combination of these antibodies was used in a direct panning technique for high‐yield enrichment of normal B lymphocytes from peripheral blood. The enriched B cells could be further purified by lysis of T cells (final yield, on average 72 ± 8% of initial B cells) or by a second panning (yield, 35 ± 11%). The purified B cells contained <1% contaminating T cells and <0.5% monocytes and were used in an assay for B‐cell‐stimulating factor which they showed a normal and very reproducible proliferative response.

Serological cloning of cancer/testis antigens expressed in prostate cancer using cDNA phage surface display

Serological cloning of tumor-associated antigens (TAAs) using patient autoantibodies and tumor cDNA expression libraries (SEREX) has identified a wide array of tumor proteins eliciting B-cell responses in patients. However, alternative cloning strategies with the possibility of high throughput analysis of patient sera and tumor libraries may be of interest. We explored the pJuFo phage surface display system, allowing display of recombinant tumor proteins on the surface of M13 filamentous phage, for cloning of TAAs in prostate cancer (PC). Control experiments established that after a few rounds of selection on immobilized specific IgG, a high degree of enrichment of seroreactive clones was achieved. With an increasing number of selection rounds, a higher yield of positive clones was offset by an apparent loss of diversity in the repertoire of selected clones. Using autologous patient serum IgG in a combined biopanning and immunoscreening approach, we identified 13 different TAAs. Three of these (NY-ESO-1, Lage-1, and Xage-1) were known members of the cancer/testis family of TAAs, and one other protein had previously been isolated by SEREX in cancer types other than PC. Specific IgG responses against NY-ESO-1 were found in sera from 4/20 patients with hormone refractory PC, against Lage-1 in 3/20, and Xage-1 in 1/20. No reactivity against the remaining proteins was detected in other PC patients, and none of the TAAs reacted with serum from healthy subjects. The results demonstrate that phage surface display combined with postselection immunoscreening is suitable for cloning a diverse repertoire of TAAs from tumor tissue cDNA libraries. Furthermore, candidate TAAs for vaccine development of PC were identified.

Wnt expression and canonical Wnt signaling in human bone marrow B lymphopoiesis

BackgroundThe early B lymphopoiesis in mammals is regulated through close interactions with stromal cells and components of the intracellular matrix in the bone marrow (BM) microenvironment. Although B lymphopoiesis has been studied for decades, the factors that are implicated in this process, both autocrine and paracrine, are inadequately explored. Wnt signaling is known to be involved in embryonic development and growth regulation of tissues and cancer. Wnt molecules are produced in the BM, and we here ask whether canonical Wnt signaling has a role in regulating human BM B lymphopoiesis.ResultsExamination of the mRNA expression pattern of Wnt ligands, Fzd receptors and Wnt antagonists revealed that BM B progenitor cells and stromal cells express a set of ligands and receptors available for induction of Wnt signaling as well as antagonists for fine tuning of this signaling. Furthermore, different B progenitor maturation stages showed differential expression of Wnt receptors and co-receptors, β-catenin, plakoglobin, LEF-1 and TCF-4 mRNAs, suggesting canonical Wnt signaling as a regulator of early B lymphopoiesis. Exogenous Wnt3A induced stabilization and nuclear accumulation of β-catenin in primary lineage restricted B progenitor cells. Also, Wnt3A inhibited B lymphopoiesis of CD133+CD10- hematopoietic progenitor cells and CD10+ B progenitor cells in coculture assays using a supportive layer of stromal cells. This effect was blocked by the Wnt antagonists sFRP1 or Dkk1. Examination of early events in the coculture showed that Wnt3A inhibits cell division of B progenitor cells.ConclusionThese results indicate that canonical Wnt signaling is involved in human BM B lymphopoiesis where it acts as a negative regulator of cell proliferation in a direct or stroma dependent manner.

Immunomagnetic isolation of NK and LAK cells.

The present study describes the immunomagnetic isolation of human natural killer (NK) and lymphokine activated killer (LAK) cells. Antibodies against CD56 and sheep anti-mouse IgG-coated magnetic monodisperse particles (Dynabeads M-450) were used for the positive isolation of CD56+ cells from unstimulated mononuclear cells (PBMC). A highly enriched population of CD56+ cells (less than or equal to 3% contaminating cells) was obtained with this method. The cellular yield of CD56+ cells was high (5.3% of the unseparated PBMC). The CD56+ cells remained unactivated after separation and preserved their functional characteristics, as measured by cytotoxic activity against the NK sensitive K562 cells. Incubating the CD56+ cells with IL-2 resulted in high LAK activity, as measured by cytotoxic activity against Daudi cells. Large numbers of functionally active CD56+ cells were obtained from IL-2 stimulated lymphocytes using anti-CD56 coated Dynabeads 450. A further enrichment of effector cells with LAK activity was accomplished by depleting the CD56+ cells for T-cells by anti-CD3 coated Dynabeads M450. The immunomagnetic isolation technique described was easy to perform, did not require expensive equipment and yielded NK and LAK cells of satisfactory purity.

Wnt3A activates canonical Wnt signalling in acute lymphoblastic leukaemia (ALL) cells and inhibits the proliferation of B-ALL cell lines

Acute lymphoblastic leukaemia (ALL) is the most common malignancy in children. Recently, there has been a growing interest in Wnt signalling in several aspects of cellular development, including cancer formation. Little is known about Wnt signalling in B‐ALL. We investigated whether activation of canonical Wnt signalling could occur in B‐ALL cells and thereby play a potential role in cellular growth and/or survival. This study found that Wnt3A induced β‐catenin accumulation in both primary B‐ALL cells and B‐ALL leukaemia cell lines. Further, Wnt3A was shown to induce nuclear translocation of β‐catenin and TCF/Lef‐1 dependent transcriptions in the B‐ALL cell line Nalm‐6. Examination of the mRNA expression pattern of WNT ligands, FZD receptors and WNT antagonists in Nalm‐6 cells identified a set of ligands and receptors available for signalling, as well as antagonists potentially available for modulating the response. Functional analyses showed that Wnt3A inhibited the proliferation of several, but not all, B‐ALL cell lines studied. Finally, microarray analysis was used to identify several Wnt3A target genes involved in a diverse range of cellular activities, which are potential mediators of the Wnt3A‐restrained proliferation.

Characterization of a promoter region supporting transcription of a novel human β-galactoside α-2,6-sialyltransferase transcript in HepG2 cells

Abstract In humans, two transcripts encoding β-galactoside α-2,6-sialyltransferase (EC 2.4.99.1.) have previously been described. One of the transcripts is widely expressed, whereas the other is restricted to mature B-cells. In this study we demonstrate the existence of a third transcript in the hepatoma cell-line HepG2. The expression of this transcript is controlled by a promoter region which efficiently supports transcription in HepG2 cells, and which harbours putative binding sites for liver-enriched and acute phase inducible transcription factors.

Human B-Cell Neoplasms in Relation to Normal B-Cell Differentiation and Maturation Processes

Publisher Summary This chapter reviews the functional studies on human neoplasias particularly emphasizing on B-cell neoplasms, thus limiting to the B-cell compartment. The diagnostic systems in human oncology are based on the relationship between neoplastic cells and their normal counterparts. The progress in immunology, at the basic level as well as at the clinical methodological level, has allowed reassessment to take place with regard to neoplasms associated with lymphoid tissues. Such studies have shown that among non-Hodgkin lymphomas (NHL) and chronic lymphocytic leukemia (CLL), the great majority of neoplasms have B-cell characteristics. The only known function of the B-cell compartment is the production of antibodies. Thus, in all the aspects discussed in the chapter, emphasis has been on Ig-related phenomena. However, the chapter also discusses biological processes taking place in the B-cell compartment. These include a number of fascinating phenomena such as generation of diversity, allogeneic exclusion, isotype light-chain restriction, clonal “abortion”, isotype coexpression and switch, B-cell proliferation, affinity maturation, and antibody production and secretion.