Cell biology of human IgM-producing hybridomas derived from a fusion of human spleen lymphocytes with mouse myeloma cells

Hybrids were derived from the fusion of mouse myeloma cells with human spleen cells from a patient with active idiopathic thrombocytopenia. Of 288 initially seeded cultures, 186 were found to produce human Ig. The growth and Ig production rates, cloning efficiencies using different feeder layers and the karyotype were determined for 9 clones that stably produced human monoclonal IgM (2-100 micrograms/ml) for at least 9 months. All cells of the Ig-producing hybridoma clones were positive for cytoplasmic-Ig, whereas only 20-65% of cells expressed surface Ig (mu and chains). Human monoclonal antibodies in mass cultures were derived in serum-free PRMI 1640 medium. Two clones produced human IgM (nearly 2 mg/ml) in the ascitic fluid of nude mice. Feeder cells of peritoneal macrophages from Balb/c mice enabled more efficient recloning of human x mouse hybrids than did thymocytes. Nearly all subclones derived from 2 clones were found to produce the same monoclonal antibodies as the parental lines. Information on the individual parameters of a hybridoma cell line may be helpful in the large-scale production of human monoclonal antibodies.     

Positive selection of mouse B and T lymphocytes and analysis of isolated populations by flow cytometry

Mouse spleen cells were separated into Ig+ and Ig- populations by positive selection using petri plates coated with rabbit anti-mouse immunoglobulin. The Ig- cells were subsequently incubated with mouse monoclonal alloantisera to Thy1.2 prior to a second positive selection. The adherent populations were characterized as B (Ig+) or T (Thy1.2+) lymphocytes on the basis of surface immunofluorescence and mitogen-induced proliferation. Analysis of the 2 isolated populations by flow cytometry showed that B and T lymphocytes could be distinguished by their forward light scatter as well as their fluorescence after incubation with fluorescein diacetate.     

Separation of lymphocyte subpopulations in carp Cyprinus carpio L. by monoclonal antibodies: immunohistochemical studies.

Lymphoid cell populations in various organs of the carp Cyprinus carpio L. were investigated using a series of mouse monoclonal antibodies raised against carp thymocytes and carp serum Ig. Clones have been designated as Ig+T+, Ig+T- or Ig-T+ on the basis of the reactivity with thymocytes and serum Ig in the enzyme-linked immunosorbent assay (ELISA) screening. Their reaction to the lymphoid organs of carp was investigated on cryostat sections and cytocentrifuge slides using immunoperoxidase techniques. IG+T+ clones could be subdivided into those that stained reticular fibres around blood vessels in various organs (R+) and those that did not (R-). The former stained most thymocytes and most peripheral lymphocytes as well as plasma cells whereas the latter did not stain cortical thymocytes and some peripheral lymphocytes. IG+T- clones were negative for thymocytes but positive for plasma cells and a certain population of peripheral lymphocytes. Ig-T+ clones reacted similarly to Ig+T+R- clones. It is concluded that fish lymphoid cell populations can be distinguished based upon differences in cell surface and/or cytoplasmic determinants. The monoclonal antibodies described can be used for further structural analysis of the determinants and for functional separation of T- and B-like cells in the 'lower' vertebrates.     

Soluble anti-mu monoclonal antibodies prime resting B cells to secrete immunoglobulins in response to interleukins-4 and -5.

Soluble anti-immunoglobulin (Ig) antibodies have been generally found to inhibit Ig secretion in B cells, via largely unknown mechanisms. To investigate this phenomenon further a two-step culture system was used in which B cells are primed for 24-72 h with various soluble monoclonal or polyclonal anti-Ig antibodies: after washing the cells were placed in readout cultures with a combination of interleukin (IL)-5 and IL-4. Using this protocol B cells primed with (mitogenic or nonmitogenic) anti-mu monoclonal antibodies differentiated into large numbers of IgM-secreting cells, comparable to responses to lipopolysaccharide. In contrast, priming with polyclonal rabbit anti-Ig or monoclonal anti-kappa antibodies, markedly inhibited Ig secretion induced by IL-4 + IL-5. In addition, anti-mu was markedly inhibitory if left in the readout cultures with the two lymphokines. These results, therefore, indicate that appropriate cross-linking of surface IgM receptors on B cells can prime the cells to secrete Ig when they are restimulated by T cell-derived lymphokines in the absence of anti-mu. In contrast co-ligation of both surface IgM and surface IgD receptors apparently results in powerful inhibition of Ig secretion, which is not reversed by stimulation with IL-4 plus IL-5.     

Immunoglobulin bearing blood leucocytes in the Pacific hagfish

The occurrence of immunoglobulin (Ig) bearing leucocytes in the blood of the Pacific hagfish, $\text{Eptatretus}$$\text{stoutii}$, was examined using a murine monoclonal antibody (45.3) and a rabbit antiserum specific for hagfish serum Ig. Binding of antibody 45.3 to hagfish leucocytes assessed by radioimmunoassay was inhibited by preincubation of antibody with purified serum Ig thus verifying the presence of cell surface Ig cross reactive with serum Ig. The monoclonal antibody identified approximately 65% of blood leucocytes as Ig+ve while the rabbit antiserum indicated 81% Ig+ve cells. Both antibody preparations failed to react specifically with cells from mouse, horned shark, tunicate or sea star; this indicates the distinctive nature of hagfish Ig. The high percentage of blood cells bearing surface Ig in the hagfish raises the possibility that lymphocyte divergence to separate B and T pathways may not have occurred in this most primitive vertebrate. Alternatively, an Ig-like specificity characteristic of both “T” and “B” lymphocytes may have been detected. In any event, a subset of Ig negative leucocytes is evident in hagfish.     

Monoclonal anti-idiotype antibodies against the murine B cell lymphoma 38C13: characterization and use as probes for the biology of the tumor in vivo and in vitro

To establish a murine model for the monoclonal anti-idiotype immunotherapy of B cell lymphoma, a panel of rat and murine monoclonal anti-idiotype antibodies of several different isotypes was generated against the surface immunoglobulin of the murine B cell tumor 38C13 (38C). Xenogeneic antibodies were made from fusions of rat spleen cells immunized with the 38C idiotype. Syngeneic monoclonal anti-idiotypes were generated from mice immunized with the idiotype conjugated to the protein carrier KLH. Small differences were noted in the ability of the antibodies to cross-block one another, but all appeared to be directed against the same or closely spaced idiotopes on the immunoglobulin molecule. The antibodies selectively precipitated surface Ig from 38C tumor cells and not from normal mouse spleen cells. They were used to selectively stain 38C tumor cells in cell suspensions for FACS analysis or immunohistochemical staining of tissue sections from mice bearing the tumor. As the malignancy progressed, the number of tumor cells found in all tissues examined increased. Thus, the anti-Id antibodies provided a specific probe for tumor cell detection. The antibodies had no detectable effect on cell growth in vitro; however, they did cause the rapid transient loss of the expression of cell surface Ig. This modulation was concentration and time dependent but not 100% complete. Re-expression of the Id occurred by 24 h following removal of the anti-Id antibodies. When these antibodies were used in sensitive radioisotope and enzyme linked immunoassays, the tumor cells were found to secrete small amounts of idiotype in vitro and in vivo. The level of idiotype detected in vivo correlated with tumor growth and inversely with survival. This work is an attempt to develop further an animal model system in which to test the diagnostic and therapeutic effects of monoclonal anti-idiotype antibodies.     

Surface immunoglobulins in chronic lymphatic leukaemia, macroglobulinaemia and myelomatosis

The direct method of immunofluorescence was applied for the detection of surface immunoglobulins (Ig) on lymphocytes, obtained from bone marrow and peripheral blood, especially from patients with paraproteinaemia. The results in chronic lymphatic leukaemia (CLL), in the macroglobulinaemia of Waldenström (MW), and in multiple myeloma (MM) show three different patterns as far as the relationship between cytoplasmic and membrane Ig is concerned. In CLL a monoclonal proliferation of cells with surface Ig was found, but the percentage of cells with cytoplasmic fluorescence was normal. In MW the further differentiation in Ig-secreting cells seems to be intact. In MM the percentage of lymphocytes in the peripheral blood with membrane-bound Ig was within normal limits or decreased.     

In Vitro Production of Monoclonal and Polyclonal Immunoglobulins by Peripheral Blood Mononuclear Cells in Human Plasma Cell Myeloma

The in vitro monoclonal and polyclonal immunoglobulin (Ig) production of peripheral blood mononuclcar cells was studied in human multiple myeloma (four IgG myelomas, one IgA myeloma) and in one patient with benign monoclonal gammopathy. Using an enzyme-linked immunosorbent assay with anti-class-specific antisera and antisera against idiotypic structures on the myeloma protein, it was possible to quantilate separately monoclonal and polyctonal Ig of the same class in cell culture supernatants. After stimulation with pokcweed mitogen (PWM) patients' cells produced lower amounts of polyclonal Ig than cells from healthy adults. In contrast, production of monoclonal Ig could not be enhanced by PWM. Moreover, the kinetics of monoclonal Ig production was different from that of polyclonal Ig. Myeloma cells contained large amounts of monoclonal Ig while their content of polyclonal Ig was low. A rapid release of preformed monoclonal Ig during the first day of culture was not inhibited by puromycin. A later phase of release was partly suppressed by puromycin and was probably caused by active protein synthesis.     

Some chronic lymphocytic leukemia cells bearing surface immunoglobulins share determinants with T cells.

One set of antigens is common to some chronic lymphocytic leukemia (CLL) cells bearing surface immunoglobulins (Ig) and normal T cells. Proliferating cells from thirty-eight patients were studied with four antisera recognizing normal human T but not B cells. These antisera were raised in rabbits against (a) Sezary cells, (b) blood lymphocytes from a patient with sex-linked agammaglobulinemia, (c) T lymphoblasts and (d) thymus cells. In four CLL cases, the cells expressed the receptor for sheep erythrocytes and lacked surface Ig. Cells from thirty-four CLL cases bore monoclonal surface Ig and did not bind sheep erythrocytes. Twelve out of these thirty-four cases of CLL had cells which were lysed by one or, more frequently, by the four anti-human T cell xenoantisera. By absorption experiments, one set of at least three antigens common to the cells of some of these CLL and T cells was defined. Depending on the patient, the cells can either carry one, some or all of the antigens of this set. However, it was also demonstrated by absorption that these cells lacked antigens particular to the T cell lineage, while the cells from T CLL cases carried both sets of antigens.     

Phorbol ester-induced differentiation of chronic B lymphocytic leukaemia cells--regulatory impact of autologous and allogeneic accessory cells.

Phorbol ester (TPA) induction of chronic lymphocytic leukaemia (CLL) cells can be used as a model system for the study of human B cell differentiation. We have analysed the role of accessory cells and the correlation to target cell surface Ig phenotype in TPA-induced morphological and functional differentiation of 12 CLL populations. A more than three-fold increase in secreted IgM was seen in 10 of 12 cases, with the strongest responses in patients having monoclonal serum IgM. CLL populations negative for or having only weak surface mu chain expression were less inducible. The impact of autologous and allogeneic accessory cells on TPA induction was studied in cell enrichment/depletion experiments using both physical and cytotoxic antibody techniques. CLL cells physically depleted of autologous E+ and monocytic (light density fraction) cells still responded to TPA. This response could be enhanced by allogeneic E- light fraction cells. Further depletion of autologous accessory cells by treatment of the E- high density fraction CLL cells with a panel of monoclonal antibodies plus complement demonstrated the permissive role of one or two populations of autologous cells expressing low avidity E receptors and the T3, T4 and T8 antigens. Augmenting T cells of similar phenotype were found among allogeneic cells from normal individuals. Thus, TPA-induced IgM secretion in biopsy B-CLL cells is regulated by minute numbers of autologous helper T cells. Furthermore, the Ig secretory response of CLL populations seems to be correlated with the surface Ig the surface immunoglobulin phenotype of the leukaemic cells.     

Characterization of monoclonal antibody specific to the Z39Ig protein, a member of immunoglobulin superfamily

Z39Ig, a recently identified immunoglobulin (Ig) superfamily member, is localized in the pericentromeric region of human chromosome X and detectable in all human tissue, but it is predominantly expressed in fetal human tissues as well as in adult lungs and placenta. In the present study, we generated a monoclonal antibody against Z39Ig protein to investigate the immunological role of Z39Ig protein on various immune cells. The anti-Z39Ig mAb that we generated specifically bound to Z39Ig protein on human promonocytic THP-1 cells, monocytes isolated from human peripheral blood mononuclear cells (PBMC) and mature CD14(+) dendritic cells (DC) differentiated from umbilical-cord blood CD34(+) hematopoietic progenitor cells. In addition, a signal through the Z39Ig protein induced an obvious cell surface expression of HLA-DR on THP-1 cells mediated by MHC class II transactivator (CIITA). These data suggest that the Z39Ig protein might be a critical molecule to regulate an immune response mediated by phagocytosis and/or antigen presentation.     

Variants of lymphoid lines produced with ricin A-chain monoclonal antibody conjugates

Conjugates of ricin A-chain with monoclonal anti-light chain antibodies specifically killed cells hearing kappa or lambda immunoglobulin (Ig) light chains. Exposure of cells from B-lymphoblastoid cell lines (B-LCL) to conjugate for less than 30 h had only a slight effect on cell growth, but on 48 h exposure a marked killing effect was achieved. After recovery of growth, cells were re-exposed to conjugate for 9-14 days. Treatment of cells from the EB4 line (sIgG lambda) in this way yielded 4 variants which showed a marked reduction in levels of surface Ig lambda and secreted Ig lambda with slight, or no, reduction in MHC class II expression and similar growth rates to the parent line. Variant lines retained their phenotype over long periods of culture.     

Particle-labeled antibodies. I. Anti T-cell antibodies attached to plastic beads by poly-L-lysine.

A simple and inexpensive method for the detection of T-cell surface antigens is introduced using polyacrylic plastic beads (PAA-beads) as indicator particles. The beads are easily visible in ordinary light microscope and make the method convenient for routine laboratory use, also in laboratories possessing no special equipment.The method is an indirect test using a first, absorbed rabbit anti-T serum to sensitize the cells and a second, sandwich antiserum (goat anti-rabbit Ig) coupled to the indicator beads. Instead of glutaraldehyde used by other investigators to attach antibodies to the beads, we used poly-l-lysine which did not affect antibody titers.The F(ab)₂ fragment of the sandwich goat anti-rabbit Ig antibody was employed to avoid binding of antibody-coated beads to Fc-receptor-bearing cells. Sensitivity, specificity and reproducibility of the method were tested on murine and human lymphoid cells treated with the respective anti-T serum. The data obtained show that the sensitivity of the method depends on: (a) the bead concentration, (b) the concentration of F(ab)₂ fragment of the second antiserum used to coat the beads, and (c) the concentration of specific antibody used to sensitize the cells. Specificity and reproducibility of the method were found to be good. The percentage of positive cells with this method are in good agreement with those reported by means of well established immunological methods.     

Lymphocyte binding of aggregated IgG and surface Ig staining in chronic lymphocytic leukaemia

Eleven selected patients with chronic lymphocytic leukaemia were evaluated for lymphocyte binding of aggregated IgG and surface Ig staining in order to classify them into B and T cell types. Ten of the eleven patients bound aggregates and stained for surface Ig. In the individual ten patients the number of cells binding aggregates was high (88–100%, mean 96%) whereas the number staining for surface Ig was more variable (8–100%, mean 62%). Parallel and double labelling experiments with aggregates and sheep red blood cell rosettes, a human T cell marker, provided evidence that aggregates were binding to B cells only, even when surface Ig was not detectable. Aggregates did not bind to human thymocytes. Evidence was presented that lymphocytes from some cases of CLL have low but not absent amounts of surface Ig that may be only partially detected by fluorescence techniques. Aggregate binding appears to be a more sensitive method for the detection of B lymphocytes than surface Ig staining.In one of the eleven patients the leukaemic cells were negative in the aggregate binding test. Separate studies on this case also indicated an absence of surface Ig staining and a high percentage of cells forming sheep red blood cell rosettes. It would appear that this case represented a T cell leukaemia.     

Human donor bone marrow cells induce in vitro "suppressor T cells" that functionally suppress autologous B cells

We have reported a beneficial effect of donor vertebral body bone marrow cells (DBMC) infusions in cadaver renal allograft recipients in a 6-year follow-up, but with a transient increase in early (6 month) postoperative CMV infections and concomitant suppressed immunoglobulins (Ig) production. We also found that although there was no difference between the DBMC-infused and non-infused (control) groups in the development of donor-specific antibody, we now describe an additional difference seen in the percent reactive antibody (PRA) reactivity against a panel of HLA antigens that developed postoperatively. We hypothesize that (allogeneic) antigen presenting cells in the DBMC, systemically infused, caused the generation of recipient T suppressor (T4-suppressor) cells, thereby "inducing" a negative influence on B cell Ig production. We tested this notion in vitro by incubating PBL from CMV IgG positive laboratory volunteers with either (allogeneic) T-cell depleted DBMC or donor spleen cells (DSPC) from (the same) cadaver donors. After 7 days, the (responding) T cells were collected using magnetic beads and placed in culture with purified B cells freshly obtained from the same (autologous) CMV positive volunteer. To these cultures were added either media or 40 ng of CMV antigen. After 3, 5, 7, and 9 days, the expression of surface anti-CMV Ig was measured by flow cytometry using a panel of fluorescent markers double-labeled for activated B cells (CD20, CD19, and HLA DRw) and CMV-FITC. We also determined the phenotype of the cultured T cells using anti-CD3, CD4, and CD62L specific monoclonal antibodies. B cells that had been in contact with autologous T cells derived from DBMC cultures (TBM) were less likely to express anti-CMV surface Ig than those cultured with DSPC (TSP). The flow cytometry analysis revealed an increase in the number of T4 suppressor cells (CD3+, CD4+, CD62L+) in the TBM group, whereas the T4 helper phenotype (CD3+, CD4+, CD62L-) predominated in the TSP group. These in vitro findings support the notion that (allogeneic) DBMC infusions can induce a T4 suppressor (regulatory) influence and thereby indirectly affect B-cell function.     

Detection and characterization of monoclonal antibodies specific to IgE receptors on human lymphocytes by flow cytometry

BALB/c mice were immunized with human lymphoblastoid cells (RPMI 8866 cells) expressing surface receptors for IgE (Fc epsilon R). Spleen cells from animals displaying high titres of anti-Fc epsilon R antibodies were fused with HGPRT-deficient NSI myeloma cells. Anti-Fc epsilon R antibodies were identified by a flow cytometric assay based on their ability to block the binding of IgE-coated fluorescent latex particles to Fc epsilon R-positive cells. Fourteen monoclonal hybridoma cell lines secreting antibody of the required specificity were amplified in tissue culture and then grown in the peritoneal cavity of BALB/c mice in order to obtain ascitic fluids with high antibody titres. The specificity of each monoclonal antibody (Mab) to lymphocyte Fc epsilon R was shown by the following observations: (i) the intact monoclonal antibody molecule or, in some cases, its F(ab')2 fragments blocked the binding of IgE to several Fc epsilon R(+) cell lines different from that employed for the initial immunization; (ii) the Mab bound directly to all the Fc epsilon R(+) cell lines tested, but not to several Fc epsilon R(-) cells as determined by indirect immunofluorescence; (iii) the binding of Mab to Fc epsilon R(+) cells was selectively blocked by IgE, but not by the other classes of Ig; and (iv) Mab had no effect on the binding of IgG to Fc gamma R on normal human peripheral blood mononuclear cells (PBMC).     

A rapid solid-phase enzyme-linked binding assay for screening monoclonal antibodies to cell surface antigens

A solid-phase enzyme-linked binding assay is described for screening monoclonal antibodies to cell surface antigens. E. coli β-galactosidase was coupled to rabbit anti-rat Ig and used to detect the binding of rat monoclonal antibodies to cells which had been fixed to the wells of microtitre plates using a combination of poly-l-lysine and glutaraldehyde. This method was found to be advantageous for the large scale screening of monoclonal antibodies with a panel of cell types, and has been useful in the selection of antibodies which would be candidates for differentiation markers within the human and mouse haemopoietic systems.     

Detection and quantitation of rotavirus using monoclonal antibody coupled red blood cells: comparison with ELISA

A total of 125 faecal extracts from infants were tested by reverse passive haemagglutination (RPH) using red cells coated with a monoclonal antibody against the major group-specific rotavirus antigen (VP 6). Results were compared with those obtained using a rabbit anti-rotavirus capture, guinea pig anti-rotavirus detector-based ELISA. The specificity of the assay was confirmed by use of 'normal' immunoglobulin coupled red cells and by inhibition with rabbit antiserum. The antibody-coated red cells could be stabilised by treatment with glutaraldehyde and subsequent freeze-drying with no detectable loss of activity even after storage at 45 degrees C for 4 wk. Good correlation was obtained between RPH and ELISA. Purified bovine rotavirus could be detected by RPH down to approximately 10(5) particles in a 25 microliters vol. Similar results were obtained with polyclonal antibody coupled cells and an ELISA using monoclonal antibody. Experiments using subgroup-specific monoclonal antibodies indicated the feasibility of rapid subgroup determination.     

In vivo and in vitro assessment of B-B cell interactions: inhibition of proliferation and antibody production of the CRIA B cells mediated by the surface immunoglobulins of anti-CRIA B cells

In this work we have assessed the effect of cell surface anti-immunoglobulin (Ig) of anti-idiotypic B cells on their idiotypic counterparts in vivo and in vitro, as a surrogate for soluble anti-surface Ig, using the well-characterized anti-arsonate system. The response of A/J mice against the hapten arsonate coupled to keyhole limpet hemocyanin (ARS-KLH) is dominated by a closely related family of antibodies sharing the same determinant, named the CRIA idiotype. We show herein that a massive induction of anti-CRIA B cells, subsequent to immunization with the mAb 3665 (CRIA⁺ , arsonate binding) coupled to KLH, mediated a strong and long-lasting inhibition of this dominant oligoclonal response to arsonate. The titer of anti-arsonate antibodies remained, however, unchanged. Adoptive transfers to x-irradiated syngeneic mice showed that anti-CRIA-producing B cells have a direct effect on induction of inhibition. This was supported by the in vitro data where irradiated anti-CRIA B cells could induce inhibition of both antibody production and mitogenesis of their counterparts, CRIA B cells. This inhibitory effect could be decreased when the surface anti-surface Ig were hidden by the 3665 Fab fragments but not by anti-MHC class II antibodies. These interactions between CRIA and anti-CRIA B cells were solely Igh restricted and the inhibition was likely initiated by hyperaggregation of surface Ig. The presence of ARS-KLH-primed T cells in vitro could prevent the growth inhibition but not the suppression of antibody production. A similar profile was noticed in vitro for soluble polyclonal rabbit anti-CRIA Ab. All together, our data suggest that a negative signaling in B cells may be initiated by surface Ig of their idiotypic partners subsequent to a strong cross-linking of their surface Ig receptors.