Levels of expression of CD19 and CD20 in chronic B cell leukaemias.

AIMS: To investigate whether the antigen levels of the B cell lineage markers CD19 and CD20 can distinguish between normal and neoplastic B cells or characterise distinct expression patterns among the chronic B cell leukaemias. METHODS: Peripheral blood cells from 70 patients with B cell disorders and 17 healthy donors were analysed by quantitative flow cytometry. Direct immunofluorescence staining was performed with phycoerythrin conjugated CD19 and CD20 monoclonal antibodies. Standard microbeads with different capacities to bind mouse immunoglobulins were used to convert the mean fluorescence intensity (MFI) values into number of antigen molecules/cell, expressed as antibody binding capacity (ABC). RESULTS: CD19 and CD20 ABC values in leukaemic B cells differed from those of normal blood B lymphocytes. The results identified distinct profiles of CD19 and CD20 expression in the various types of B cell leukaemias. In all leukaemias studied except hairy cell leukaemia (HCL), CD19 expression was significantly lower than the mean (SD) value in normal B cells (22 (7) x 10(3) molecules/cell), as follows: chronic lymphocytic leukaemia (CLL), 13 (7) x 10(3); B prolymphocytic leukaemia (B-PLL), 16 (9) x 10(3); splenic lymphoma with villous lymphocytes (SLVL), 15 (11) x 10(3); mantle cell lymphoma (MCL), 10 (7) x 10(3). In HCL there was strong CD19 expression (38 (16) x 10(3)). In contrast, the level of expression of membrane CD20 was higher than the mean (SD) value in normal B cells (94 (16) x 10(3) molecules/cell) in MCL (123 (51) x 10(3)); B-PLL (129 (47) x 10(3)); SLVL (167 (72) x 10(3)); and HCL (312 (110) x 10(3)); while it was significantly lower (65 (11) x 10(3)) in CLL compared with normal B cells and the other B cell leukaemias. CONCLUSIONS: Quantitative determination of CD19 and CD20 may provide useful diagnostic information for the study of B lymphoproliferative disorders.     

Generation of non-MHC restricted killing in cultures stimulated with B cells from chronic lymphocytic leukaemia patients: phenotypic characterization of the precursor and effector cells.

Freshly isolated B cells from chronic lymphocytic leukaemia patients (B-CLL) have been previously shown to induce a strong proliferative response and high levels of NK-like activity in lymphocytes from healthy donors. The present paper deals with the origin, mitotic state, target spectrum and cell surface phenotype of the NK-like effectors generated after stimulation with B-CLL. Experiments using large granular lymphocytes (LGL) and T cells as responders demonstrated that most of the precursors of the newly generated NK-like effectors express the CD3 antigen. The induction of NK-like activity paralleled cell activation, as judged by blast transformation, thymidine uptake and appearance of cell surface activation markers. The newly generated NK-like effectors displayed a T cell phenotype and a broader target repertoire than native NK cells.     

Separation of T lymphocytes from normal individuals and patients with B lymphocyte chronic lymphocytic leukaemia.

Previous studies have shown that lymphocytes from patients with chronic lymphocytic leukaemia have a diminished response to mitogens which stimulate T cells. Chronic lymphocytic leukaemia is most often a disease of accumulating B cells so that T lymphocytes are diluted by large numbers of leukaemic cells. Direct comparison with the responses of normal lymphocytes to mitogenic stimulation is therefore suspect. To circumvent this difficulty, a method of isolating T cells from normal individuals and patients with chronic lymphocytic leukaemia was developed. Lymphocytes containing an average of 16.1 per cent B cells from normal individuals were applied to IgG-anti-IgG-coated Degalan bead columns and held at 4 degrees for 2 hours. The eluted cells contained less than 2 per cent B cells. When chronic lymphocytic leukaemic lymphocytes, containing an average of 68.6 per cent B cells, were applied to IgG-anti-IgG columns, the eluted cells contained 36.4 per cent B cells. To improve the purification of T lymphocytes, columns of uncoated Degalan beads were used to remove non-specifically adherent cells. Eluted lymphocytes were then applied to IgG-anti-IgG columns. This resulted in the recovery of purified populations of T cells with less than 2 per cent contamination with B cells. Patients with chronic lymphocytic leukaemia were found to have lymphocytes with either a normal density or a low density of surface immunoglobulins. B cells were successfully removed from lymphocyte suspensions in all cases of chronic lymphocytic leukaemia with a normal density of lymphocyte surface immunoglobulins. In the three cases of chronic lymphocytic leukaemia with low density surface immunoglobulins, separation by this method was unsuccessful. However, an enriched T-cell population was obtained when leukaemic lymphocytes which had lost all detectable surface immunoglobulins were passed through a column coated with heat-aggregated IgG.     

Immunoglobulin heavy chain variable region gene utilization by B cell hybridomas derived from rheumatoid synovial tissue.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that primarily affects synovial joints. Activated B lymphocytes and plasma cells are present in the synovial tissue and are thought to contribute to the immunopathology of the rheumatoid joint. To investigate rheumatoid synovial B lymphocytes, we have generated B cell hybridomas from synovial tissue of an RA patient. Here we describe the immunoglobulin VH gene repertoire of eight IgM- and 10 IgG-secreting synovial-derived hybridomas. The VH4 gene family is highly represented (38.5%) in this panel of hybridomas compared with the frequency of VH4 gene expression in circulating B lymphocytes reported previously (19-22%) and with the VH4 gene frequency we observed in a panel of hybridomas derived in the same manner from the spleen and tonsil of normal individuals (19%). The increased frequency of VH4 gene expression was not due to the expansion of a single B cell clone in vivo as none of these hybridomas was clonally related. Two synovial-derived hybridomas secreted autoantibodies; one (VH3+) secreted an IgM-rheumatoid factor (RF) and the other (VH4+) secreted IgM with polyreactive binding to cytoskeletal proteins and cardiolipin. The antibodies secreted by the remaining synovial-derived hybridomas were not reactive with the autoantigens tested. The VH gene usage in a proportion (5/17) of synovial-derived hybridomas that expressed CD5 antigen provided preliminary evidence that CD5+ B cells in RA synovium have a similar increase of VH4 gene expression reported for CD5+ B cells from normal individuals and patients with chronic lymphocytic leukaemia.     

Leukaemic phase of mantle zone (intermediate) lymphoma: its characterisation in 11 cases

Sixteen patients presented with B cell leukaemia (white cell count 26-269 x 10(9)/l) which could not be classified as chronic lymphocytic (CLL), prolymphocytic leukaemia, or follicular lymphoma in leukaemic phase. Eleven patients (10 men, one woman) corresponded histologically to intermediate (INT) or mantle zone lymphoma, and five, with less well defined features, were designated small lymphocytic lymphoma with cleaved cells. The blood films showed a pleomorphic picture with lymphoid cells of predominantly medium size with nuclear irregularities and clefts. The membrane phenotype of the circulating cells showed strong immunoglobulin staining and reactivity with CD5 and FMC7 in all cases tested; CD10 was positive in six out of nine cases. The membrane phenotype of two of the five cases of small lymphocytic lymphoma was close to those of B-CLL and three resembled INT lymphoma. Bone marrow trephine biopsy specimens showed a diffuse pattern of infiltration in INT lymphoma. The median survival of these patients was less than two years, suggesting that a leukaemic presentation is associated with poor prognosis. By combining data from histology, membrane markers, and peripheral blood morphology, the leukaemic phase of typical INT lymphoma can be defined in most cases.     

Comparison of some lymphocyte markers in B-cell chronic lymphocytic leukaemia and systemic lupus erythematosus (the B lymphocyte subset)

Spontaneous mouse red-cell rosette formation (M-rosette), Leu 1 (CD 5) monoclonal antibody binding, ecto-nucleotidase enzyme reactions and mitogen responses of peripheral blood lymphocytes in 10 patients with B-cell chronic lymphocytic leukaemia (CLL), in 8 with systemic lupus erythematosus (SLE) and in 10 controls were studied. The numbers of Leu 1 (CD 5) positive B lymphocytes in CLL and SLE were higher than in controls. These are called "autoregulatory" B lymphocytes, which are thought to be independent of T-cell regulatory effects. A significant increase of mouse rosette forming cells (MRFCs) was found in CLL, while in SLE and in controls their number was low. Compared to the B lymphocytes of SLE patients and controls, those of CLL patients showed diminished responses to pokeweed mitogen stimulation parallel to their decreased level of 5'-nucleotidase. Correlations between these markers and the features of isolated M-rosette positive CLL B lymphocytes are discussed.     

Human lymphocyte markers defined by antibodies derived from somatic cell hybrids. II. A hybridoma secreting antibody against an antigen expressed by human B and null lymphocytes.

A hybridoma (FMC4) has been derived which secretes antibody showing selective reaction with human B lymphocytes, monocytes and some null lymphocytes. Few, if any, T lymphocytes in normal blood are stained, although stimulation of lymphocytes with PHA leads to an increase in the proportion of cells reacting with the hybridoma antibody. The antibody reacts with B and null lymphoblastoid cell lines but not with T cell lines. B chronic lymphocytic leukaemia (CLL) cells but not T-CLLs are stained and null-type acute lymphoblastic leukaemia (ALL) cells but not T-type ALL also react. Normal blood myeloid cells do not react with FMC4 supernatant whilst some myeloid leukaemias do. The expression of the antigen reacting with FMC4 supernatant suggests that FMC4 may secrete an antibody against the human equivalent of the Ia antigen.     

Antisera against leukaemia-associated antigens on human lymphocytes.

Antisera were raised in rabbits against leukaemic lymphosarcoma (LSL) cells which carried surface markers of both thymus-derived T lymphocytes (T cells) and bone marrow-derived B lymphocytes (B cells). After absorption with leucocytes, erythrocytes and serum proteins from normal individuals, the antisera demonstrated significant complement-dependent cytotoxicity against leukaemic cells from patients with acute lymphoblastic leukaemia (ALL) (9/11), LSL (7/9) and chronic lymphocytic leukaemia (CLL) (9/12), with an antibody titre of 1:64 or greater. The antisera did not react with: (a) blood lymphocytes from clinically healthy individuals (0/23), patients with ono-lymphoproliferative disorders (0/8) and normal umbilical cords (0/3), (b) normal lymphocytes stimulated by pokeweed mitogen (0/7), allogeneic lymphocytes (0/3), fetuin (0/1), purified protein derivative (PPD) (0/2), and candida antigen (0/1); (C) normal marrow cells (0/3), (D) normal thymocytes (0/2) and (E) leukaemic cells from patients with acute myeloblastic (AML) (0/10) and chronic granulocytic leukaemia (CGL) (0/3). However, the antisera did react with lymphoblastoid cells from continuous B-cell lines derived from an AML patient and from a non-leukaemic individual and, to a lesser extent, with lymphocytes from patients with infectious mononucleosis. The antisera also reacted with lymphocytes from chronically infected tonsils. Cytotoxicity of the antisera against lymphoblastoid and tonsillar cells was inhibited by ALL and CLL cell-lysates; and, conversely, cytotoxicity against ALL cells was inhibited by the lymphoblastoid cell extract. In contrast, a cell lysate or extract from normal inhibited by the lymphoblastoid cell extract. In contrast, a cell lysate or extract from normal lymphocytes did not inhibit cytotoxicity toward lymphoblastoid, tonsillar or ALL cells. Cytotoxicity of the antisera was neutralized by a goat anti-rabbit IgG (GAR IgG). These results suggest that the antisera contained antibodies reactive with antigens possibly common to neoplastic lymphocytes, tonsillar cells, lymphoblastoid cells and some lymphocytes from patients with infectious mononucleosis.     

Immunologic phenotype of lymphocytes in chronic B-cell lymphocytic leukemia. I. B- and T-lymphocytes in chronic lymphocytic leukemia]

This article contains the review and the critical discussion of studies of T and B lymphocytes in chronic B-cell lymphocytic leukaemia. It is not explicitly known now, which stage of differentiation of lympho-haemopoietic cells undergoes malignant transformation in CLL-B. Basing on VDJ recombination activity it is supposed that some cases derive from the stem cell, others--from B lineage cells.     

EBV Infection Induces Expression of the Transcription Factors ATF-2/c-Jun in B Lymphocytes but not in B-CLL Cells

B cell type chronic lymphocytic leukaemia (B-CLL) cells carry the Epstein-Barr virus (EBV) receptor CD21 and can be infected in vitro with the virus. The infected cells exhibit an unusual EBV program, they express the nuclear proteins but not latent membrane protein 1 (LMP-1). Similar cells were encountered in lymphoid tissues of infectious mononucleosis (IM) patients and in lymphoproliferations of immunosuppressed patients. EBV infected B-CLL cells can be regarded as model for this viral program. In B cells the regulation of LMP-1 is executed mainly by EBV encoded nuclear antigen 2 (EBNA-2), interacting with several cellular proteins and these complexes bind to specific sequences in the LMP-1 promoter. ATF2 and c-Jun were shown to be among the interacting partners of EBNA-2. These molecules can be detected in experimentally infected B lymphocytes. We found c-Jun and/or phosphorylated ATF-2 (p-ATF-2) expression in some B-CLL ex vivo samples. They disappeared or their expression declined promptly in explanted cells, even if they were infected with EBV in vitro. Activation of the infected B-CLL cells by exposure to CD40L was accompanied by p-ATF-2 and c-Jun but not by LMP-1 expression. In one of three clones tested, subsequent treatment with histone deacetylase inhibitors (HDACi), TSA or n-butyrate, could induce LMP-1. Treatment with phorbol-12, 13-dibutyrate (PDB) induced LMP-1 expression in three of four clones. Neither the HDACi nor the PDB treated cells survived.     

Soluble forms of CD21 and CD23 antigens in the serum in B cell chronic lymphocytic leukaemia

By using pairs of monoclonal antibodies (MAbs) to different epitopes on CD21 and CD23 antigens, it has been shown that both antigens are readily detectable in cell-free supernates of cultures of B cells expressing these antigens on the cell surface. The antigens remained in the soluble fraction after high speed centrifugation. Sera from normal individuals contained significant amounts of CD21 antigen, whereas little CD23 antigen was detectable. By contrast CD23 but not CD21 antigen was present in urine. Sera from patients with B cell chronic lymphocytic leukaemia (B-CLL) contained increased amounts of both antigens. The levels were related to the surface expression of antigen on the leukaemic cells and the number of cells in the blood. The possible functional role of soluble forms of B cell antigens and the diagnostic potential of their detection in body fluids are discussed.     

The thiol-proteindisulfide oxidoreductase in human mononuclear cells of blood and bone marrow

The in vivo function of the thiol-proteindisulfide oxidoreductase (TPO, EC 1.8.4.2; proteindisulfide isomerase, EC 5.3.4.1) in biosynthesis of immunoglobulin was investigated by studying the enzyme content in human lymphoid and other cells by an immunocytochemical method. In contrast to peripheral blood, B lymphocytes which showed no or no demonstrable TPO, normal as well as malignant bone marrow plasma cells (all Ig classes) were found to contain abundant amounts of this enzyme. TPO containing plasma cells were identified by double-staining techniques. This finding suggests that TPO is involved in the terminal step of B cell differentiation and immunoglobulin biosynthesis. Besides plasma cells, approximately 10% of mononuclear marrow cells as yet unidentified medium-sized and large cells, exhibited also strong anti-TPO reactivity. Furthermore, using surface-cytoplasmic double staining methods, monocytes from human peripheral blood could be identified to represent the only cytoplasmic TPO-containing normal mononuclear blood cells.     

Phenotypic heterogeneity of B cell chronic lymphocytic leukaemia

The peripheral blood lymphocytes from 39 patients from the Latvian S.S.R.T., U.S.S.R. with chronic lymphocytic leukaemia (CLL) have been phenotyped with various monoclonal antibodies representing the major clusters of differentiation (CD) used for phenotyping B cells. A clear delineation of two groups of patients was evidenced. The major group (33/39) possessed leukaemic cells bearing surface immunoglobulins (SIg) at a low density, Class II HLA, and CD5, CD24 and CD37 molecules but not CD21, CD22 and CD35. CD23 antigen was seen only once under microscope examination, but could be visualized by flow cytometry. CD6 antibody reacted with cells from about 1/3 of this group of patients. In the six patients of the second group the leukaemic phenotype was SIg+, Class II HLA+, CD5+, 24+, 37+, 21+, 22+, 35+, 23+ and 6-. The main finding is the concomitant expression of CD22, CD21 (CR2) and CD35 (CR1) molecules, all involved in B cell activation. It is not yet known whether these observations correlate with different clinical evolutions of the disease.     

Unique Immunological Behaviour of CD8+ (Suppressor T Cell) Leukaemia Cells

A patient with chronic lymphocytic leukaemia and elevated serum IgM antibody is described. A multiparameter surface marker analysis of his peripheral blood lymphocytes demonstrated a unique phenotype of OKT3-, OKT11+, E-rosetting+, OKT4-, OKT8+, OKIa1+, OKCLL+, OKDR+, Tac+, characterizing the disease as suppressor (CD8+) T cell chronic lymphocytic leukaemia. Because of the uncommon phenotype and the abnormal serum immunoglobulin pattern, in vitro functional assays were performed that showed decreased mitogenic response to the phytohaemagglutinin, but increased response to concanavalin A. However, these leukaemic T cells demonstrated phytohaemagglutinin-specific suppressor cell activity on normal blood lymphocytes. In vitro functional studies indicated that a defect in the patient's T cells may cause IgM hypergammaglobulinaemia. The regulatory function of the patient's T cells on immunoglobulin synthesis by normal B cells was found to be mediated by a soluble factor secreted from neoplastic T cells.     

Beta-glucuronidase activity of lymph node imprints from malignant lymphomas and chronic lymphocytic leukaemia.

beta-Glucuronidase activity was semiquantitatively estimated in the cells of lymph node (LN) imprints from patients with Hodgkin's disease (HD), diffuse non-Hodgkin's lymphomas, chronic lymphocytic leukaemia (CLL), normal lymph nodes, and benign lymphadenopathies. In addition, in some of these cases beta-glucuronidase activity was semiquantitatively determined in peripheral blood smear lymphocytes. The beta-glucuronidase score (betaGS) was very low in the cells of the LN imprints from patients with diffuse non-Hodgkin's lymphomas. The LN lymphocytes of HD had a normal betaGS independently of the histological subtype of the disease, while in the LN imprint of CLL the enzyme activity was low, normal, or high. The betaGS of the lymphocytes in LN imprints of normal controls and HD were in general significantly lower compared with he lymphocytes of the peripheral blood smears in the same cases. The relation of our findings to the B and T cell origin of malignant lymphomas and chronic lymphocytic leukaemia is discussed.     

Cell surface localized IgM-kappa immunoglobulin reactivity in a case of chronic lymphocytic leukaemia

The cell surface of peripheral lymphocytes from a patient with chronic lymphocytic leukaemia reacted strongly with fluorescein conjugated anti-IgM and kappa serum. Moreover in the presence of complement anti-IgM serum was cytotoxic. This patient was unusually resistant to various therapeutic measures. Serum immunoglobulin levels were slightly subnormal. The reactivity described differs from other chronic lymphocytic leukaemias studied but could be found in a number of Burkitt cases. The leukaemia cells may represent the malignant transformation of a normal lymphoid cell specialized to carry immunoglobulin on its cell membrane.     

Human B cells express membrane-bound and soluble forms of the CD14 myeloid antigen.

The expression of the myeloid differentiation antigen CD14 on the B lineage was analyzed. A CD14-specific monoclonal antibody was used to isolate the antigen from normal B, B-type chronic lymphocytic leukemia cells, and a representative Epstein-Barr virus-transformed B lymphoblastoid cell line (EBVLCL). A soluble form of this protein was detected in the culture supernatant of all the B cell types tested. The molecule expressed in the normal B and B-type chronic lymphocytic leukemia cells was identical in size to the 52,000 mol. wt monocyte-isolated CD14 glycoprotein. A 64,000 mol. wt antigen was isolated from the lymphoblastoid cell line. Similar 2-D gel electrophoretic patterns to that of the monocyte-derived CD14 were obtained from the normal B and B-type chronic lymphocytic leukemia cell-isolated molecules. These similarities were reflected in minor isoelectric point (pI) differences between the polypeptide spots (pI 4.8), in the first dimension, and identical molecular weight (52,000) in the second dimension. The EBVLCL-isolated polypeptide, when analyzed by 2-D gel electrophoresis, showed a pI identical to that of the myeloid antigen (pI 4.6). The isolated soluble form was of smaller (47,000 mol. wt, normal B and B-type chronic lymphocytic leukemia cells) or similar size (64,000 mol. wt, lymphoblastoid cell line) compared with their corresponding membrane-bound forms. Interestingly, two-colour immunofluorescence analysis showed that only two out of four CD14-specific mAb tested bound to the B cells. We conclude that the CD14 antigen is, in fact, expressed in the B lineage. Its cell surface expression and serum level in the prognosis of B-type chronic lymphocytic leukemia patients needs to be evaluated.     

Impact of cryopreservation on B cell chronic lymphocytic leukaemia phenotype.

BACKGROUND AND OBJECTIVES: Freezing is a practical approach for cell preservation for retrospective studies. The aim of this work was to check the cryopreservation impact on B cell chronic lymphocytic leukaemia phenotype. MATERIAL AND METHODS: Blood samples from 15 CLL patients were analyzed freshly and after freezing at -196 degrees C, without separation, and thawing. Results were compared by Student's paired t-test. RESULTS: The phenotype of fresh CLL cells was as follows: CD19+, CD5+, faint CD20, CD23+/-, weak CD22 and sIg, CD37+, HLA-DR+, FMC7-. After cryopreservation, the percentage of CD5 and CD23 positive cells decreased, whereas HLA-DR positive cells increased moderately. The CLL Matutes's score was modified in 6 cases out of 15 (40%). CONCLUSION: Cryopreservation modifies B cell chronic lymphocytic leukaemia phenotype, by decreasing CD5 and CD23 expression.     

Study of pregnancy-associated alpha2-glycoprotein in relation to populations of human blood leucocytes.

Pregnancy-associated alpha2-glycoprotein (alpha2-PAG) was identified by immunofluorescence on the surface membrane of mononuclear cells from normal individuals with an extensive range of plasma concentrations of the glycoprotein. The incidence of positive cells did not correlate with plasma levels but was significantly raised in chronic lymphocytic leukaemia. Anti-alpha2-PAG antibody interfered with E and Fc rosette formation but not with production of C3 rosettes. A large proportion of C3 receptor-bearing cells (B lymphocytes) but only a tiny fraction of E-rosette-forming cells (T lymphocytes) were positive for the protein. Antibody directed against human Ia antigen markedly reduced staining for alpha2-PAG.     

CD3+ CD8+ T cell lymphocytosis masking B cell leukaemia.

A patient with CD3, CD8 positive lymphocytosis presented with features consistent with T cell chronic lymphocytic leukaemia/proliferations of large granular lymphocytes. The marrow and blood lymphoid populations (19.4 x 10(9)/l) contained more than 80% CD3 and CD8 positive cells with no evidence of a monotypic B cell population. A biopsy specimen of a vasculitic rash showed a diffuse infiltrate of CD3, CD8 positive cells into the upper dermis, consistent with T cell lymphocytic disease. After follow up for two years without treatment the blood lymphocyte count was 53 x 10(9)/l and was composed of cytologically small lymphocytes. A monoclonal SIg M D k lymphoid population (more than 90%) was demonstrable in sample blood and marrow aspirate. Gene rearrangement studies carried out on DNA extracted from peripheral blood lymphocytes at presentation and at two year follow up exhibited JH and Ck immunoglobulin gene rearrangement but no rearrangement of T cell receptor TcR gamma and beta genes. It is thought that this is the first well documented case of an aggressive CD8 positive lymphocytosis preceding, or in response to, an underlying B cell neoplasm.