The HB-6, CDw75, and CD76 differentiation antigens are unique cell-surface carbohydrate determinants generated by the beta-galactoside alpha 2,6-sialyltransferase.

Expression of the beta-galactoside alpha 2,6-sialyltransferase (alpha 2,6-ST) was shown to regulate the generation of multiple cell-surface differentiation antigens (Ags) that may be necessary for lymphocyte function. A new mAb was produced, termed HB-6, that was shown to identify a novel neuraminidase-sensitive cell-surface Ag expressed by subpopulations of human lymphocytes and erythrocytes. In attempting to isolate a cDNA encoding the HB-6 antigen by expression cloning, a cDNA encoding the alpha 2,6-ST (EC 2.4.99.1) was obtained. Since expression of the alpha 2,6-ST protein was shown to be limited to the Golgi apparatus, the cell-surface HB-6 Ag was demonstrated to be the product of alpha 2,6-ST activity. Interestingly, alpha 2,6-ST expression also generated two other neuraminidase-sensitive lymphocyte cell-surface differentiation Ags, CDw75, and CD76. The HB-6, CDw75, and CD76 mAb identified distinct Ags that were differentially expressed by different B cell lines and exhibited different patterns of expression in tissue sections. These results indicate that alpha 2,6-ST expression is a critical regulatory step in the formation of the Ags that are recognized by these mAb, and that an alpha 2,6-linked sialic acid residue is an essential component of each Ag. Thus, expression of a single ST can result in the generation of multiple distinct antigenic determinants on the cell surface which can be distinguished by mAb and may have regulatory roles in lymphocyte function.

Novel type of proliferating lymphoplasmacytoid cell with a characteristic spotted immunofluorescence pattern.

We examined bone marrow from myeloma patients for the presence of cells with the characteristics of the clonogenic cell in the myeloma stem cell assay. We identified a novel type of cell that contained cytoplasmic immunoglobulin of the relevant idiotype located in a cytoplasmic spot. This "spotted" Ig could be located in the rough endoplasmic reticulum. Spotted cells are highly proliferative, as evidenced by the nuclear staining with the antibody Ki67, and were found in the bone marrow from most of the myeloma patients studied. This type of cell was also present in patients with immunocytomas, in some cases of benign monoclonal gammopathy, and in patients in the state of polyclonal hypergammaglobulinemia. IgG subclass distribution of so-called spotted cells and plasma cells, found in a patient with pseudo biclonal gammopathy, indicates that spotted cells are intermediate between B cells and plasma cells. Spotted cells express the B cell-associated antigens HB4 and HB6 but do not express other B cluster of differentiation antigens or plasmacytoid antigens tested.

Binding of the human complement subcomponent C1q to hybrid mouse monoclonal antibodies.

Activation of the classical pathway of the complement system is initiated by the binding of C1q to antibody complexes. Here we evaluated the C1q binding capacity of series of monospecific and bispecific hybrid mouse monoclonal antibodies (mAb) and compared them with parental (conventional) mAb. The hierarchy in C1q binding capacity of the bispecific anti-HuIgA1/HRP mAb with homologous H-H chain combinations (IgG2a-2a, IgG2b-2b and IgG1-1) and the parental anti-HuIgA1 or anti-HRP mAb was identical; IgG2a greater than IgG2b much greater than IgG1. Hybrid IgG1-2a mAb bind intermediate amounts of C1q when compared with the IgG1 and IgG2a parental antibodies. IgG1-2b and IgG1-1 hybrid mAb did not bind any C1q, like the IgG1 mAb. We could not observe any difference in C1q binding efficiency between monovalently bound IgG1-2a, IgG2a-2a and IgG2b-2b anti-HuIgA1 HRP mAb and the bivalently bound IgG1-2a, IgG2a-2a and IgG2b-2b anti-HuIgA1 mAb, respectively. Furthermore, these hybrid ms anti-HuIgA1 and bs anti-HRP/HuIgA1 mAb were able to lyse HuIgA1-coated erythrocytes, in the presence of 50% human serum, as efficiently as their parental counterparts. These data indicate that a simultaneous binding of both F(ab') fragment to antigen is not a necessary prerequisite for binding and activation of C1q.

Influence of cytomegalovirus infection on the recovery of humoral immunity after autologous bone marrow transplantation.

Recovery of B-cell number and function was studied in 23 patients with hematological malignancies treated with high-dose chemoradiotherapy followed by autologous bone marrow transplantation (auto-BMT) in relation to the presence or absence of cytomegalovirus (CMV) infection. B cells recovered rapidly after auto-BMT and specific antibodies to herpes viruses remained nearly unchanged. Both were independent of the CMV status of the patients. However, the capacity of peripheral blood B cells to differentiate in vitro into cytoplasmic immunoglobulin (Ig)-positive cells (plasma cells) on pokeweed mitogen stimulation in the presence of normal T-cell help was significantly better in CMV-negative patients than in CMV-positive patients after auto-BMT, but was decreased in both groups. Serum Ig levels were, in contrast, higher in CMV-positive patients than in CMV-negative patients after auto-BMT.

Indicator cell lines for the detection of hidden mycoplasma contamination, using an adenosine phosphorylase screening test.

Mycoplasmas are a major cause of cell culture contamination and are especially troublesome during HAT selection. The enzyme adenosine phosphorylase (adoP) is present in all common mycoplasma species but is considered to have a low activity in mammalian cells. However, using an adoP screening test, we have observed that some cell cultures do possess an intrinsic adoP activity leading to false positive results. Moreover, as a false negative result, we encountered a variant of Mycoplasma orale (identified after cultivation on agar and immunostaining) which was not detectable with the adoP screening in cell culture supernatants and only at low levels in cell lysates. To increase the low signal/noise adoP ratio found there, we used an indicator cell line with low intrinsic activity. Indicator cells were inoculated with the test supernatant and the adoP activity of these infected cells were measured after lysis. The procedure diminished the effect of biological variation in intrinsic enzyme activity between the several cell lines tested. Furthermore, in another mycoplasma infected cell line (with M. fermentans), this infection was only reliably detected using these indicator cells. With this procedure we obtained rapid results which were concordant with those obtained using the time consuming cultivation on agar.

Production of hybrid hybridomas based on HAT(s)-neomycin(r) double mutants.

A detailed procedure is described for the preparation of hybrid hybridomas, that produce bispecific antibodies. This is achieved by fusing two hybridoma cell lines that are phenotypically distinct (HAT(s)/neo(r) and HAT(r)/neo(s)) and thereby allow for the selection of the appropriate hybrid cells. HATs mutants were obtained from one of the two fusion partners by 8-azaguanine treatment; these mutant phenotypes were found in an unexpected high frequency. For the introduction of the dominant neo(r) marker gene in one of the HAT(s) fusion partners, a retroviral vector was used in order to obtain a high efficiency of gene transfer. Our method was very effective in the production of hybrid hybridomas, so-called quadromas. The detection of bispecific antibodies was based on simultaneous binding by one antibody of two different antigens, or on the presence of two different H chain isotypes in this molecule.

Comparison of various in vitro assays for efficacy screening of immunotoxins.

Before using immunotoxins in vivo, their efficacy is evaluated in in vitro assays. In this study we compare six different assays for the evaluation of immunotoxins: protein and DNA synthesis inhibition assay, chromium release assay, cell line colony assay, limiting dilution assay and clonogenic assay. All assays except the chromium release assay show specificity of the immunotoxins in appropriate concentrations. The protein and DNA synthesis inhibition assays are easy to perform and, therefore, suitable for initial screening, while the clonogenic assay seems to be the best one for immunotoxin efficacy determination.

Mitogenic stimulation of malignant B cells CLL: diminished in-vitro stimulation with anti-CR1 antibodies.

Peripheral B lymphocytes of five CLL patients were tested in a radioimmunoassay to determine the density of the C3b receptor (CR1) and the cells were assayed for their ability to mature into IgM secreting cells after in-vitro culture with a combination of Pokeweed Mitogen (PWM) and antibodies directed against CR1. Despite the presence of normal amounts of CR1 on the leukemic B cells, crosslinking of these receptors by anti-CR1 antibodies stimulated only a fraction of the leukemic cell population to differentiate into IgM secreting cells. These results add to the partial functional impairment of CLL-B cells.

Putative myeloma precursor cells expressing 2,6 sialic acid-modified antigens actually belong to the erythroid lineage.

The Golgi enzyme alpha2,6-sialyltransferase modifies glycoconjugates by adding sialic acid. In lymphocytes, different epitopes that result from this modification have been identified by the B cell-related CDw75, CDw76, HB4 or HB6 Ab. We previously described positive staining with these Ab of a highly transferrin receptor-positive (CD71) cell type in the bone marrow of multiple myeloma patients. These cells were distinct from plasma cells, but did contain Ig of the same isotype and idiotype as seen in the plasma cells. We postulated a precursor role for this cell type in myeloma. Here, we report that this CD71+ (HB4/HB6/CDw75/CDw76)+ cell is an erythroid precursor cell instead. RT-PCR did not detect Ig mRNA, and from immuno electron microscopy Ig appeared to be endocytosed rather than synthesized by these cells. At their cell surface the erythroid/megakaryocytic markers CD36 and CD41, and the erythroid-specific glycophorin A can be detected, while haemoglobin can be detected antigenically in the cytoplasm. Finally, purified cells proliferate in vitro upon addition of erythropoietin. Uptake of Ig could be explained by the presence of Fc gammaRIII(CD16), which has also been found on other haematopoietic precursor cells.

Fluorescence in situ hybridization analysis shows the frequent occurrence of 14q32.3 rearrangements with involvement of immunoglobulin switch regions in myeloma cell lines.

In many B-cell malignancies, 14q32.3 chromosomal rearrangements involving the immunoglobulin heavy chain (IgH) locus have been shown to be pathognomonic for the disease. Although in myeloma heterogeneous and complex karyotypes are found, 14q32.3 translocations are prominent. However, owing to the telomeric position of the IgH locus, 14q32.3 translocations may be easily missed. We established fluorescence in situ hybridization (FISH) assays on chromosomes and DNA fibers to determine both the occurrence of 14q32.3 rearrangements in myeloma cell lines and the precise localization of the breakpoints in the IgH locus. Our results show that 14q32.3 chromosomal rearrangements are present in almost every myeloma cell line analyzed (17 of 19, 89%). Breakpoint analysis of the lines harboring one or more 14q32.3 rearrangements with the use of fiber-FISH revealed the involvement of switch regions in the IgH locus in 11 of 17 cell lines. Remarkably, pseudogamma genes without switch regions were involved in 3 of 17 cell lines, all derived from IgA myelomas. Three of 17 cell lines contained breakpoints outside a switch or immunoglobulin heavy chain constant region. The almost ubiquitous presence of 14q32.3 rearrangements suggests an obligatory role in the development of myeloma. The high incidence of breakpoints involving switch regions indicates an oncogenic event in a late stage of B-cell differentiation.

Multiple Ig classes on rabbit B lymphocytes.

Rabbit PBL were studied regarding the presence of different classes of s-Ig, under experimental conditions, ensuring the endogeneous origin of these proteins. About 40% of the lymphocytes are B cells (Fab positive and a1 positive in a1 homozygous rabbits). IgG positive lymphocytes could be found, but only using and anti dll conjugate (allotype located on the Fd gamma region of IgG). Anti Fc gamma conjugates were negative. Most of the B cells are IgM positive, most of these IgM cells however were also positive for either IgG or IgA. Np lymphocytes were found bearing both IgG and IgA. Careful analysis of the percentages of various isotypes found on B cells suggests that some "IgD" positive lymphocytes could be present. Results are discussed in relation to B cell differentiation.

Enrichment and selection of hybrid hybridomas by Percoll density gradient centrifugation and fluorescent-activated cell sorting.

Hybrid hybridomas, producing bi-specific monoclonal antibodies that react with horseradish peroxidase and human IgA1 were isolated by sorting the double-fluorescent cells on single-cell basis after fusion of two hybridomas, previously labelled green or red by octadecylamine-FITC or -TRITC, respectively. The double-fluorescent fused cells were significantly different in AXL (size) and RAS (internal structure) distribution compared with the (non-fused) mono-fluorescent cells. The percentage of double-fluorescent cells and the viability of these cells could be increased by Percoll density gradient centrifugation. As a result, there was an 8-fold increase of total isolated hybrid hybridomas (up to 30% of all tested clones) compared to isolations without Percoll density gradient centrifugation. All the isolated hybrid hybridoma clones had similar amounts of DNA, equal to the sum of the DNA of both parental hybridomas.

Characterization of the colony-forming cell in monoclonal gammopathies.

The nature of the colony-forming cell in the bone marrow of patients with monoclonal gammopathy, as defined in the stem cell assay described by Hamburger and Salmon, has been studied. It could be shown that the colony-forming cells produce immunoglobulins of the same idiotype, heavy chain and light chain, as the monoclonal bone marrow cells in the patient. Data regarding the presence or absence of J chain in the colonies, the failure to observe isotype-switch in the growing colonies, as well as the lack of inhibition of colony formation using antiidiotypic antibodies, strongly suggest that colony formation in vitro reflects proliferation of the clonogenic stem cell in the bone marrow without apparent differentiation. The stem cell may be of plasma cell nature.

Cellular expression of idiotopes defined by monoclonal antibodies.

Monoclonal anti-idiotype (Id) antibodies with specificities for determinants related to the antigen-binding sites of 3 BALB/c myeloma proteins, MOPC-460, HOPC-8 and J558, were used to study Id expression on murine lymphocytes. The monoclonal antibodies were shown to react only with Id structures associated with immunoglobulin on B cells. None of these 3 Id nor a VH Id, detected by a monoclonal antibody made against HOPC-8 heavy chain, were found on T cells. These Id were detected on splenic B cells in neonatal mice; the frequencies in normal, nude and germ-free mice were similar: MOPC-460 Id+: 1.05 +/- 1.7/10(4) spleen cells, HOPC-8 Id+: 1.45 +/- 1.2/10(4) and J558: 0.35 +/- 0.6/10(4). Almost all Id+ cells bore surface IgM, a few expressed surface IgG. MOPC-460 Id+ IgG+ cells were mainly gamma 2a+ or gamma 2b+, whereas J558 and HOPC-8 Id+ IgG+ cells were gamma 3+.

HB4 antibody recognizes a carbohydrate structure on lymphocyte surface proteins related to HB6, CDw75, and CD76 antigens.

Cells regulate the specificity of the carbohydrate chains on their membrane-bound glycoconjugates by differential expression of glycosyltransferases. In lymphocytes, beta-galactoside alpha 2,6-sialyltransferase is reportedly involved in the generation of epitopes recognized by HB6, CDw75, and CD76 mAb. The HB4 mAb binds to an Ag present on subpopulations of B and NK cells. We now show that this Ag represents another member of a set of neuraminidase-sensitive, alpha 2,6-sialyltransferase-generated sugar Ag. Transient expression of a cDNA encoding this enzyme in COS cells generated a minor population of HB4+ cells that was completely contained within the HB6+ COS cell population. Using various proteinases and an inhibitor of N-linked carbohydrate processing, we show both epitopes to represent components of N-glycosylated membrane proteins. Remarkably, porcine thyroglobulin, an alpha 2,6-NeuAc+ glycoprotein, is specifically recognized by both mAb. These data underline a close relationship between HB4 and HB6 epitopes and imply further that both mAb react with oligosaccharide chains irrespective of the carrier molecule nature. Thus, the terminal sugar residue sialic acid plays a pivotal role in at least four distinct epitopes that are expressed differentially in immune cells. This may point at an important role for these epitopes in biologic recognition.

Determination of the plasma cell labelling index with bromodeoxyuridine in a double fluorescence technique.

A new technique is described in which plasma cells actively synthesizing DNA can be identified in a heterogeneous cell population such as bone marrow. This method uses bromodeoxyuridine (BrdU) and a fluorescent monoclonal antibody against BrdU in combination with cytoplasmic staining for immunoglobulin (Ig). In 26 patients with monoclonal gammopathy (MG) the plasma cell labelling index (LI) as determined by this immunofluorescent method was compared with the tritiated thymidine (3H-TdR) LI. No difference in sensitivity was found between the two methods. As the determination of the plasma cell LI with the BrdU/anti BrdU method is easy to perform and results can be obtained within 4 h, this immunofluorescent method seems to be an attractive alternative to the laborious time-consuming classic 3H-TdR LI.

Human monocyte-mediated cytotoxicity towards erythrocytes induced by hybrid mouse monoclonal antibodies: effect of antibody binding valency on IgG-Fc gamma R interaction.

In this study, we describe the ability of hybrid mouse monoclonal antibody (mAb) to induce monocyte-mediated cytotoxicity towards human IgA1-coated E (HuIgA1-E), and the effect of mAb binding valency on Fc gamma RI-mediated ADCC. All hybrid monospecific (ms) anti-HuIgA1 and bispecific (bs) anti-HuIgA1/HRP mAb were capable of inducing monocyte-mediated lysis of HuIgA1-E, in spite of differences in mAb densities essential for optimal lysis. The cytotoxicity induced by hybrid mAb which consist of one or more mIgG2a H chains was predominantly mediated via Fc gamma RI, as shown by inhibition studies on monocytes with Fc gamma RI-blocking mAb TB-3 (approximately 80% inhibition). However, partial inhibition of mIgG1-2a and mIgG2a-2b-induced cytotoxicity (20-50%) was observed by using Fc gamma RII-blocking mAb IV.3 or CIKM5. For hybrid mIgG1-1 mAb the opposite was true; the cytotoxicity was predominantly mediated via Fc gamma RII (70-80%) and less via Fc gamma RI (20-30%). Comparing the hybrid ms anti-HuIgA1 mAb-induced cytotoxicity with the cytotoxicity induced by hybrid bs anti-HuIgA1/HRP mAb of the same isotype, we observed a decrease in cytotoxicity towards HuIgA1-E sensitized with univalently bound bs anti-HuIgA1/HRP mAb. This decrease was only found for Fc gamma RI-mediated ADCC (mIgG2a-2a, mIgG1-2a and mIgG2a-2b). This diminished recognition of univalently bound IgG relative to bivalently bound IgG by Fc gamma RI was also observed with U937 effector cells. In conclusion, this work shows that hybrid mAb are able to induce monocyte-mediated cytotoxicity towards E-HuIgA1 and that there appears to be an effect of Ag-IgG binding valency on Fc gamma RI-mediated cytotoxicity.

Fc receptors on rabbit lymphocytes. Identification and organ distribution of rosette-forming cells; cocapping with surface immunoglobulin.

Fc receptor (FcR)-bearing cells were demonstrated using ox erythrocytes coated with homologous IgG-type antibodies (EA gamma) in rabbit peripheral blood leukocytes (PBL) and in various lymphoid organs. Discrimination of the rosette-forming cells (RFC) is carried out after prior ingestion of tetramethylrhodamine isothiocyanate-labeled latex particles and in transmission electron microscopic studies. Most of the nonlymphoid cells (5-10%) in PBL and spleen cell suspensions expose FcR. These nonlymphoid cells are almost absent in other lymphoid organs, except in bone marrow. The average percentage of cells rosetting with IgG-sensitized erythrocytes (EA gamma RFC) in lymphoid cell preparations of the various tissues was as follows: PBL 25%, bone marrow 65%, appendix 37%, spleen 40%, Peyer's patches 44%, thymus 2% and peripheral lymph node 27%. The nature of FcR-bearing PBL was further studied using F (ab')2 anti-IgM, anti-IgA or anti-T cell conjugates. About half of the population of B cells, bearing IgM or IgA express FcR. Moreover, about 80% of the RFC are found within the B cell population. Only a few T cells were found rosetting with EA gamma suggesting that most of the non-B lymphoid RFC are "null" cells. In different lymphoid organs, the percentages of EA gamma RFC and B cells are comparable but not identical A greater part of the EA gamma RFC also expresses the receptor for the third component of complement. After capping of membrane IgM determinants, FcR is located in the same cap on the majority (60%) of the FcR-positive IgM-capped cells.

Efficacy of an anti-CD138 immunotoxin and doxorubicin on drug-resistant and drug-sensitive myeloma cells.

Multidrug resistance is an increasing problem in the treatment of cancer. We evaluated in vitro the effect of an anti-CD138 plasma-cell-specific immunotoxin (IT, B-B4-SO6) in combination with the chemotherapeutic drug doxorubicin on drug-sensitive and drug-resistant variants of the multiple-myeloma (MM)-derived cell line RPMI8226 and freshly isolated malignant-myeloma cells. Drug-resistant RPMI8226 cells were still sensitive to the IT, although to a lesser extent than drug-sensitive cells. In the clonogenic assay, using 10 nM B-B4-SO6, at least 5 logs kill was found for drug-sensitive RPMI8226 cells, vs. 2.5 logs kill for the drug-resistant RPMI8226 cells. When a sub-optimal dose of 1 nM IT was combined with 3 ng/ml doxorubicin, which was toxic for drug-sensitive but not for drug-resistant cells, an additive effect was found for drug-sensitive RPMI8226 cells. The IT did not influence the sensitivity of resistant cells for doxorubicin. We therefore speculate that this type of IT, may be of more value in combination with primary chemotherapy. The effect of B-B4-SO6 on malignant-myeloma cells of patients was investigated in a viability assay. Both drug-sensitive and drug-resistant cells from MM patients were sensitive to B-B4-SO6. After 2 days, a 50% kill of malignant cells was found when 10 nM IT were used. Doxorubicin was effective only on sensitive cells, and there was a tendency for an additive effect in the combination of these cells.