Wim P. Zeijlemaker

Immunoperoxidase procedures to detect monoclonal antibodies against cell surface antigens. Quantitation of binding and staining of individual cells

An immunoperoxidase method has been developed which allows accurate and sensitive quantitation of the binding of monoclonal antibodies to cell surface antigens. Monolayers of fixed cells were prepared in wells of Terasaki micro-test plates and monoclonal antibodies bound to cell surface antigens were identified by the unlabeled antibody-enzyme method of Sternberger (1974). The cell-bound peroxidase could either be quantified per well or visualized on individual cells by the use of appropriate substrates for peroxidase. Experimental procedures are described in detail and results obtained with several monoclonal antibodies with specificity for different target cells are shown. Limitations and applications of the technique are discussed.

Effects of IL-4, IL-5, and IL-6 on growth and immunoglobulin production of Epstein-Barr virus-infected human B cells

In the present study we investigated whether interleukin-4 (IL-4), IL-5, and IL-6 could enhance the efficiency of Epstein-Barr virus (EBV) transformation for the generation of specific human monoclonal antibody (HuMAb)-producing B-cell lines directed against erythrocyte Rhesus(D) antigen. In newly EBV-infected B cells, IL-4 and IL-6 caused a comparable enhancement of proliferation and of total IgG and IgA production. IL-6 showed a much stronger effect than IL-4 on IgM production, whereas IL-4 was unique in inducing IgE production. No stimulatory effects of IL-5 on either growth or Ig production were observed. Although addition of IL-6 resulted during the early phase after EBV infection in high numbers of Ag-specific antibody-producing wells, this did not result in an increased number of stable HuMAb-secreting cell lines. When the effects of cytokines were tested on established polyclonal EBV B cells, in a high cell density culture system, only IL-6 was able to enhance Ig secretion, while no effect could be demonstrated on proliferation. These studies substantiate that IL-6 is an important regulator of proliferation and Ig production, and that it acts at distinct stages after EBV infection, but does not increase the final overall recovery of Ag-specific EBV B-cell lines.

A Human Monoclonal IgG1λ Anti‐Hepatitis B Surface Antibody

Peripheral blood mononuclear cells from a donor with a high litre of anti‐hepatitis B surface (HBs) antibodies were fused with a cell line that was positive for Epstein‐Barr virus nuclear antigen and sensitive to hypoxanthine‐aminopterine‐thymidine. A cell line was established that produces a monoclonal IgG1λ anti‐HBs antibody. Afterwards, it appeared that the anti‐HBs antibody‐producing cell line had arisen from Epstein‐Barr virus transformation of the donor B cells. The cell line is capable of producing up to 60 μg/ml of the monoclonal antibody, which has a high avidity for HBs antigen (Ag) and recognizes both ad and ay subtypes. The antibody is useful as a reagent for the detection of HBsAg in human serum. Over 1000 patient sera have been tested with a conventional third‐generation assay in parallel, and only a single discrepant serum was found.

Cooperative effects in mitogen- and antigen-induced responses of human peripheral blood lymphocyte subpopulations.

Human peripheral blood lymphocytes were separated into T cell-enriched and T cell-depleted fractions by E rosette sedimentation. These two fractions, as well as the unseparated lymphocyte suspension, were tested for their responsiveness to the mitogens phytohemagglutinin (PHA), concanavalin A (ConA) and pokeweed mitogen (PWM) and to the antigens PPD (purified protein derivative of tuberculin) and tetanus toxoid. The response to PHA, ConA and the antigens was found to be confined to the purified T cell fraction; PWM could stimulate both purified T and non-T cells. However, the T cell response to ConA, PPD and tetanus toxoid was always decreased by 50-70%, when compared to the unseparated lymphocytes. Addition of monocytes could restore the T cell response. In the response to PHA and tetanus toxoid, the (primarily unresponsive) non-T cell fraction could be recruited into proliferation by gamma-irradiated T cells. Moreover, in the response to tetanus toxoid, lymphocytes (T as well as non-T) from a nonimmune individual could be recruited into proliferation by gamma-irradiated immune T cells.

Density separation of spleen cells increases fusion frequency and yield of Ig-producing hybridomas.

The efficiency of hybridoma formation and growth after cell fusion can be much improved by fractionation of the mouse splenocytes. A simple procedure is described in which splenocytes with a specific gravity of more than 1.065 g/cm3 are selected by centrifugation on a Percoll gradient. The resulting cell suspension is largely depleted of macrophages and fibroblasts while the cell viability is improved. In fusion experiments performed with these cells, overgrowth of hybridomas by macrophages, fibroblasts and P-cells is avoided. The fusion efficiency and the frequency of immunoglobulin-secreting hybridomas is increased compared with fusions carried out with unfractionated spleen cells.

Human Cytotoxic Lymphocytes after Alloimmunization in vitro

The development of short-term cell culture systems has greatJy contributed to our insight into cell-mediated immunity. It has become apparent that after in vivo immunization, lymphoid cells develop which are, as effector cells, capable, without the help of antibody or complement, of destroying tissue culture target cells which carry the same antigen(s) to which the donor of the lymphoid cells has been immunized. Such cell-mediated cytotoxicity has been demonstrated in allograft immunity, graft versus host (GVH) reactions, delayed type hypersensitivity, autoimmunity and tumor immunity. More recently, it has become apparent that in vitro immunization of normal lymphoid cells to certain antigens can also occur when the cells are co-cultured with certain stimulator cells which carry these antigens. Such in vitro immunized effector cells display a similar cytotoxicity on target cells carrying antigens in common with the stimulator cells. This article is confined to our studies with human peripheral blood lymphocytes, in relation to the development of in vitro cytotoxicity to alloantigens after immunization in vitro. Hirschhom et al. (1965) were the first to report that human lymphocytes, co-cultured with allogeneic fibroblasts, developed a cytotoxic potential towards these cells. When cells of a human lymphoid cell line are being used as stimulators in a mixed lymphocyte culture (MLC) and also as target cells in a subsequent cytotoxicity assay, a similar target cell destruction is observed

Xenohybridization of Epstein‐Barr Virus‐Transformed Cells for the Production of Human Monoclonal Antibodies

Transformation of human B lymphocytes, obtained from hyperimmune donors with Epstein‐Barr virus, yields polyclonal cell populations in which a minority of cells produce IgG antibodies of predetermined specificity, whereas the majority of cells produce ‘non‐specific’ immunoglobulin (mainly of the IgM class). Such lymphoblastoid cell lines can be easily propagated in high‐densily cultures. Because cloning at l cell per well is not possible, stabilization of lymphoblastoid cell lines by limiting dilution is not feasible and most newly established lines cease to produce specific antibody within a few weeks. Xenohybrids, resulting from fusion of Epstein‐Barr virus‐transformed cells with NS1 mouse plasmacytoma cells, can be cloned at 1 cell per well. Stable xenohybridoma subclones, producing antibody of [he desired specificity, can be isolated after a series of limiting dilutions. In a model system, we have studied the efficiency of xenohybridization of human lymphoblastoid cells. Using this system, we have constructed IgG anti‐tetanus‐toxoid‐ and IgG anti‐HBsAg‐producing cell lines. Next, we investigated whether transformation with Epstein–Barr virus is essential in such a two‐step procedure or whether a polyclonal stimulator, such as pokeweed mitogen, could also be used. It was found that antibody‐producing xenohybrids can be obtained after stimulation with pokeweed mitogen. However, this latter system is subject to more variations and lacks the advantage of pre‐selection of antibody‐producing cells as compared to xenohybridization after transformation.

Colony-forming cells in chronic granulocytic leukemia--II. Analysis of membrane markers.

Membrane markers and functional properties in vitro of blast cells from the peripheral blood of 2 patients with chronic granulocytic leukemia were studied. Buffy-coat cells were enriched for colony-forming cells by density centrifugation (d less than or equal to 1.062 g cm-3). Upon culture, a large proportion of the (cryopreserved) low-density cells from both patients formed hemopoietic colonies that were heterogeneous with respect to size and cellular composition. Expression of membrane markers on the cells, which had the morphology of undifferentiated blasts, was studied using flow cytometry with a panel of monoclonal antibodies. A striking heterogeneity was observed in that variable numbers of cells were found to express myelomonocytic, megakaryocytic and erythroid membrane markers. Antigenic properties of colony-forming cells were studied by sorting of cells with a fluorescence activated cell sorter. Low numbers of cells (10, 4 and 1, respectively) were sorted directly into the wells of Terasaki microtest plates. With this system, it was shown that myeloid colony-forming cells from patient 1 were exclusively present in HLA-DR-positive cell fractions. Colony formation from the level of a single sorted cell was documented. Sorting of cells labeled with anti-blood-group-H antibody showed that small erythroid colony-forming cells from patient 2 were blood-group-H antigen-positive. These cells did not express HLA-DR. The other colony-forming cells from this patient and essentially all colony-forming cells from patient 1 were HLA-DR-positive and blood-group-H-negative. Although only 2 patients were tested, our studies clearly demonstrate that low-density cell fractions from the blood of patients with CGL provide distinct advantages for the study of membrane properties of hemopoietic cells and of hemopoietic differentiation in general.

The mixed lymphocyte reaction (MLR) stimulatory capacity of human lymphocyte subpopulations

Abstract Using various cell separation techniques and combinations of these, suspensions were obtained highly enriched or depleted with respect to their content of E-rosette-forming T cells, Ig-bearing B cells, Fc-receptor-bearing cells, or monocytes. These purified populations were tested for their capacity to stimulate allogeneic cells in a mixed lymphocyte reaction (MLR). It could be demonstrated that the Ig-bearing B cells provide the strongest stimulus in the MLR.

Inhibition, by vinca alkaloids and colchicine, of antigenic modulation induced by anti-CD19 monoclonal antibodies

Several clinical trials have been reported in which monoclonal antibodies (McAb) were used for therapy of lymphoid malignancies. Such trials have shown that infusion of McAb recognizing lymphoid antigens, is well-tolerated, and leads to the coating of tumor cells and tumor regression in some patients. However, the tumoricidal capacity of a McAb is hampered by the presence of circulating free antigen, antigenic modulation, development of human anti-mouse antibodies, emergence of antigen-negative variants of tumor cells and the inadequacy of host-effector cell mechanisms. We have studied the antigenic modulation induced by immunoglobulin (Ig) heavy chain switch variants of anti-CD19 McAb. Modulation of CD19 molecules was not related to the IgG subclass of the McAb. Immunofluorescence studies on the Burkitt tumor cell line Daudi showed that CD19 molecules are internalized after incubation by anti-CD19 McAb. Next, the effect of cytoskeleton inhibitors on antigenic modulation was studied. We found that antigenic modulation on Daudi cells and on an Epstein-Barr virus-transformed B-cell line was completely inhibited by vinca alkaloids (VA) or by colchicine. Interestingly, antigenic modulation of tumor cells from a VA-resistant patient, was not inhibited by VA or colchicine. These findings provide information for the rational design of more effective clinical trials with McAb.