Bone marrow purging procedure in Burkitt lymphoma with monoclonal antibodies and complement: quantification by a liquid cell culture monitoring system.

Using B1, Y29/55 and AL2 monoclonal antibodies (MoAbs) to target Burkitt lymphoma (BL) cell lines, we defined optimal conditions to lyse, in the presence of baby rabbit complement, BL cells in excess bone marrow (BM). After the purging procedure, down to one residual BL cell in 10(6) normal ones was detectable with a liquid cell culture assay. Using a cocktail of three MoAbs, on five different cell lines were observed more than 4 log BL cell depletion in samples contaminated with 1% BL cells and only one failure of the procedure on 17 experiments. However, a sixth line was constantly resistant to the procedure.

Deoxythymidine and deoxycytidine incorporation into DNA in B leukemic cells.

Measurements of radioactive DNA after incubation of normal and B leukemic peripheral mononuclear cells, from c-ALL and CLL with labeled deoxythymidine (dTh) and deoxycytidine (dCt) showed that for dTh, incorporation into DNA was similar for normal and c-ALL cells but lower in B-CLL cells and that for dCt, incorporation was highest in c-ALL and lowest in CLL cells. These results contrast with those of dTh and dCt kinase activities; the former has been previously found elevated in c-ALL cells, and the latter is found, in the present study, similar in the three groups tested.

Human B cell development. I. Phenotypic differences of B lymphocytes in the bone marrow and peripheral lymphoid tissue.

The phenotype of B lineage cells (TdT+, pre-B, IgM+, IgD-, and IgM+,IgD+) in infant and adult human bone marrow was compared with that of B cells seen in peripheral tissues such as tonsil and blood. The range of B cell-associated antibodies used included four reagents with greater than 90% reactivity on peripheral B cells: RFB4 and To15 (both p135, corresponding to CD22), RFB6 (p140 corresponding to CD21), and Y29/55, a unique B cell-specific antibody. In addition, AL-1, an antibody with virtually no reactivity against peripheral B cells was also used. The BM cell subpopulations were heterogeneous in respect of antibody reactivity. The TdT+, pre-B and IgM+, IgD- cells were AL-1+ but did not express membrane antigens recognized by the antibodies To15, RFB4 (CD22), and RFB6 (CD21). TdT+, pre-B cells, and 50% of IgM+, IgD- BM B cells were also unreactive with antibody Y29/55, the other 50% being Y29/55+. In contrast, the IgM+,IgD+ BM B cells, like peripheral B cells, were positive with antibodies To15, RFB4, RFB6, and Y29/55, but reacted only in small numbers with AL-1. The orderly differentiation-linked display of these antigens was also suggested by the findings that normal TdT+, pre-B, and IgM+,IgD- cells expressed the To15 and RFB4 (CD22) antigens in their cytoplasm (in the Golgi region). This observation was confirmed in malignant common acute lymphoblastic and pre-B blast cells, as well as in the corresponding permanent cell lines KM3 and NALM-6. In these lines the membrane expression of To15 and RFB4 could be induced by phorbol ester during a 48 to 72 hr culture period.

Rat monoclonal antibodies. I. Rapid purification from in vitro culture supernatants.

A technique for purifying rat monoclonal antibodies quickly and efficiently from in vitro culture supernatants is described. It is based on the fact that more than 95% of rat immunoglobulins carry kappa light chains. A mouse monoclonal antibody with suitable binding affinity for rat kappa light chains is immobilized on solid support and used to purify rat immunoglobulins. Milligrams of rat monoclonal antibodies may be rapidly concentrated from culture supernatants with high recovery. Rat monoclonal antibodies expressing lambda light chains (about 5% of the total) may be purified in a similar way with an appropriate anti-rat lambda chain monoclonal antibody.

Obtention of rat monoclonal antibodies reactive with human leukaemic lymphoblasts.

The Louvain rat-rat hybridoma technology was used to analyse membrane antigens present on human leukaemic lymphoblasts. Three fusions directed against non T non B leukaemic lymphoblasts finally yielded 42 distinct, stable, and specific antibody-secreting hybridomas. The cellular and molecular specificities of these monoclonal antibodies are still under investigation. However, a striking feature of our preliminary characterization is the obtention, in single fusions, of several distinct hybridomas that secrete antibodies binding to the same membrane molecule.

Increased "in vivo" lymphocyte blastogenesis in the peripheral blood of patients with atopic dermatitis and allergic contact dermatitis.

The spontaneous 3H-thymidine (3HT) labelling of some lymphocyte subpopulations has been studied in the peripheral blood of five patients with atopic dermatitis and five with widespread allergic contact dermatitis and compared with that in 10 healthy subjects. One hour after addition of 3HT to heparinized blood, lymphocytes were separated and processed with two different rosetting techniques (E-rosette test and Active E-rosette test). The cell suspensions were cytocentrifugated and autoradiography undertaken. An increased number of 3HT labelled lymphocytes was observed in the peripheral blood of patients with dermatitis as compared to controls. These labelled lymphocytes were E-rosette-forming cells (T cells) and E-non-rosette-forming cells (non-rosette-forming T cells and non-T cells). The ratio between the labelling index (LI) of E-rosette- to the LI of non-E-rosette-forming cells was in favour of T cells in allergic contact dermatitis (ratio = 3.09) whereas in atopic dermatitis (ratio = 0.93) the DNA synthesis was relatively greater in the non-rosette-forming cells. It is suggested that this increased LI of peripheral blood lymphocytes could be related to the increased derman mononuclear cell 3HT-labelling that has been reported previously in these inflammatory skin diseases.

B-cell precursors in early chicken embryos.

The ontogeny of B-cell precursors in chicken embryos from day 3 of incubation onwards has been studied. Purified antibodies to chicken Ig L, gamma, mu, alpha chains were used in a sensitive indirect immunofluorescence assayed on fixed cell smears and wax-embedded tissue sections; the location and morphology of immunoglobulin positive (Ig+) cells were determined either in phase contrast or after histological staining. Lymphoid cells containing small amounts of cytoplasmic immunoglobulin were found in 3 day and older embryonic yolk sac, 11 and 12 day blood, 11, 12 and 13 day bursal mesenchyme. cIg+ large basophilic cells were first seen in 14 day bursal follicles. It is concluded that cells enter the embryonic bursa at different developmental stages: some appear to be uncommitted stem cells, whilst others have already commenced B-cell maturation in an extra-bursal site.

Embryonic bursa development in vitro.

An in vitro culture system for embryonic chicken bursa is described, in which development, as measured by follicle formation (site of B lymphocyte development), position of large basophilic cells (stem cells), and presence of lymphocytes and cells containing cytoplasmic immunoglobulin (B cells), was comparable to that of the bursa in vivo. Using this system, preliminary studies on B lymphocyte differentiation were made. These support the view that bursal lymphocytes develop from precursors that migrate through the surrounding mesenchyme and into the follicles, but indicate that at least some of these cells may be "pre-B cells" rather than pluripotent stem cells.