Normal cellular counterparts of B cell chronic lymphocytic leukemia

In an attempt to compare B cell chronic lymphocytic leukemia (B-CLL) with its normal cellular counterpart, the cell surface phenotype of 100 cases of B-CLL was determined by using a panel of monoclonal antibodies (MoAbs) directed against B cell-restricted and -associated antigens. The majority of B-CLL cells expressed Ia, B4 (CD19), B1 (CD20), B2 (CD21), surface immunoglobulin (sIg), and T1 (CD5) but lacked C3b (CD35) receptors. In contrast, the overwhelming majority of small unstimulated B cells expressed Ia, B4, B1, B2, sIg, and C3b receptors but lacked detectable T1. Small numbers of weakly sIg+ cells could be identified in peripheral blood and tonsil that coexpressed the B1 and T1 antigens. Approximately 16% of fetal splenocytes coexpressed B1, T1, weak sIg, B2, and Ia but lacked C3b receptors and therefore closely resembled most B-CLL cells. With the phenotypic differences between the majority of small unstimulated B cells and B-CLL cells, we examined normal in vitro activated B cells and B-CLL cells for the expression of B cell-restricted and -associated activation antigens. Of 20 cases examined, virtually all expressed B5, and approximately 50% of the cases expressed interleukin-2 receptors (IL-2R) and Blast-1. Normal B cells were activated with either anti-Ig or 12-0-tetradecanoylphorbol- beta-acetate (TPA) and then were examined for coexpression of B1, T1, and the B cell activation antigens B5 and IL-2R. Only cells activated with TPA coexpressed B1 and T1 as well as B5 and IL-2R. B cells activated with either anti-Ig or TPA proliferated in the presence of IL- 2, whereas B-CLL cells did not, although they all expressed the identical 60-kilodalton proteins by immunoprecipitation. These studies are consistent with the notion that B-CLL resembles several minor subpopulations of normal B cells including a population of B cells that are activated in vitro directly through the protein kinase C pathway.     

Activation of resting human B cells by helper T-cell clone supernatant: characterization of a human B-cell-activating factor.

The effects of helper T-cell clone supernatants on resting human B cells were investigated. Four different helper T-cell clones (two T4+ and two T8+) were stimulated by anti-T3 monoclonal antibodies on Sepharose beads or anti-T11(2) plus anti-T11(3) monoclonal antibodies. The supernatants from these activated clones induced the proliferation of highly purified resting B lymphocytes from the peripheral blood. The B cells exhibited a cell size and a surface-antigen pattern (4F2 antigen and transferrin receptor) of phase G0 B cells, and they were functionally resting. In response to T-cell supernatants a large fraction of the B cells enlarged and expressed 4F2 antigens and transferrin receptors. In gel filtration, the corresponding activity migrated with an apparent Mr of 12,000-15,000. Our findings strongly support the existence of a human B-cell-activating factor acting on resting B cells and causing them to enter phase G1 of the cell cycle.     

Expression of CD5 antigen in B and T cells from umbilical cord blood, from blood of healthy adults and patients with chronic lymphocytic B-cell leukemia (PBL-B)

Antigen CD5 is the glycoprotein which belong to the scavenger receptor cysteine-rich family. Mainly there is on the T cells subpopulation. During fetal life B CD5+ cells are major subpopulation of B cells in the spleen, lymph nodes and there are also in the cord blood. In adult CD5+ cells are minor subpopulation (27%) of B cells from the peripheral blood. CD5 there are on chronic lymphocyte leukaemia B cells (B-CLL) also. Usually expression CD5 on B-CLL cells associated with weak or lack expression of the surface immunoglobulins and CD79 beta, CD20, CD22, CD21 (CR-2), CD35 (CR-1) antigens. It appeared interesting to compare the expression of CD5 antigen (the mean fluorescence intensity--MFI of CD5) on B cells from the cord blood, adults peripheral blood and B-CLL patients. MFI of CD5 on B and T cells were also compared in each groups. MFI of CD 19 was studied too. Lymphocytes from the cord blood (11 assays), adult peripheral blood of healthy volunteers (18 assays) and the peripheral blood of no treated patients with B-CLL (56 assays) were studied. The immunological phenotype of lymphocytes was evaluated with the monoclonal antibodies anti-CD5 and anti-CD19 by the flow cytometry method. We have demonstrated that MFI of CD5 on B cells from patients with B-CLL was strongest and weakest from normal individuals. MFI of CD5 on T cells from patients with B-CLL is stronger in comparison to healthy volunteers. MFI of CD19 is weakest on cells from patients with B-CLL and strongest in normal individuals. On the basis of the our results and other medical papers we suggest on the one hand that biology of B-CLL depend on deficit antigens specific for B cells lines on the other hand depend on overexpression of CD5 antigens on leukaemic B and T cells also.     

CD40-activated B-cell chronic lymphocytic leukemia cells for tumor immunotherapy: stimulation of allogeneic versus autologous T cells generates different types of effector cells

Although spontaneous remissions may rarely occur in B-cell chronic lymphocytic leukemia (B-CLL), T cells do generally not develop a clinically significant response against B-CLL cells. Because this T-cell anergy against B-CLL cells may be caused by the inability of B-CLL cells to present tumor-antigens efficiently, we examined the possibility of upregulating critical costimulatory (B7-1 and B7-2) and adhesion molecules (ICAM-1 and LFA-3) on B-CLL cells to improve antigen presentation. The stimulation of B-CLL cells via CD40 by culture on CD40L expressing feeder cells induced a strong upregulation of costimulatory and adhesion molecules and turned the B-CLL cells into efficient antigen-presenting cells (APCs). CD40-activated B-CLL (CD40-CLL) cells stimulated the proliferation of both CD4(+) and CD8(+) T cells. Interestingly, stimulation of allogeneic versus autologous T cells resulted in the expansion of different effector populations. Allogeneic CD40-CLL cells allowed for the expansion of specific CD8(+) cytolytic T cells (CTL). In marked contrast, autologous CD40-CLL cells did not induce a relevant CTL response, but rather stimulated a CD4(+), Th1-like T-cell population that expressed high levels of CD40L and released interferon-gamma in response to stimulation by CD40-CLL cells. Together, these results support the view that CD40 activation of B-CLL cells might reverse T-cell anergy against the neoplastic cell clone, although the character of the immune response depends on the major histocompatibility complex (MHC) background on which the CLL or tumor antigens are presented. These findings may have important implications for the design of cellular immunotherapies for B-CLL.     

Involvement of protein kinase C and phosphatidylinositol 3-kinase pathways in the survival of B-cell chronic lymphocytic leukemia cells

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived CD5(+) B lymphocytes. TPA (12-O-tetradecanoylphorbol 13- acetate) and interleukin-4 (IL-4) inhibit apoptosis of B-CLL lymphocytes ex vivo. We used specific inhibitors of protein kinase C (PKC), extracellular-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3-kinase) to study their involvement in TPA- and IL-4-induced survival of B-CLL lymphocytes. BisI, a specific inhibitor of PKC, induced apoptosis and inhibited the antiapoptotic activity of TPA and IL-4. B-CLL cells have a basal PKC activity that was increased by TPA but not by IL-4. TPA, but not IL-4, induced ERK activation. However, the inhibition of ERK activation did not affect the viability of B-CLL lymphocytes, demonstrating that this pathway is not involved in their survival. Inhibition of PI3-kinase by LY294002 induced apoptosis of B-CLL cells and inhibited the survival effect of IL-4 and TPA. In addition, Akt, a downstream effector of PI3-kinase activity, was phosphorylated by TPA and IL-4 in B-CLL cells, though PI3-kinase had no effect on PKC-dependent phosphorylation of Akt. Furthermore, the inhibition of PKC or PI3-kinase increased dexamethasone- and fludarabine-induced apoptosis ex vivo in the presence of survival factors. These results demonstrate that PKC and PI3-kinase are involved in the survival of B-CLL cells and suggest that inhibitors of these pathways could be combined with the drugs used in the treatment of B-CLL.     

Overexpression of cyclin D2 in chronic B-cell malignancies

Tumor progression in B-cell chronic lymphocytic leukemia (B-CLL) is thought to result from the gradual accumulation of small resting G0/G1 phase lymphoid cells rather than the proliferation of actively dividing cells. The recent identification of G1 cyclins that are likely to control both the progression through G0 and G1 phase and the G1/S transition prompted us to study the mRNA expression of D-type cyclins in the peripheral blood lymphocytes from 34 patients with B-CLL, 7 patients with lymphoplasmacytic lymphoma (LPL), and 2 patients with mantle cell lymphoma (MCL). Cyclin D2 mRNA was, on average, 5- to 10- fold overexpressed in most of the samples studied (B-CLL, 29/34; LPL, 7/7; MCL, 0/2) as compared with normal resting B lymphocytes, in which cyclin D2 mRNA was barely detectable. In situ hybridization with cyclin D2 digoxigenin-labeled mRNA probe showed that all the cells from a given sample were stained with approximately the same intensity. Cyclin D3 was never detected in any of the samples tested, whereas cyclin D1 was expressed in only the 3 cases (1 LPL and 2 MCL) bearing a t(11;14) translocation. A trisomy 12 was found in 4 of 19 (21%) B-CLL or LPL cases for which cytogenetic analysis was available. Although the cyclin D2 gene has been mapped to chromosome 12p13, there was no apparent correlation between trisomy 12 and the level of cyclin D2 expression. Cell cycle analysis by flow cytometry after staining with propidium iodide consistently showed that more than 96% of the cells were in G0/G1 phase, whatever the importance of cyclin D2 overexpression was, and that cyclin D2 overexpression in B-CLL was not associated with any modifications of the cell cycle repartition. No consistent overexpression of cyclin D2 was found in acute myeloid leukemias. In conclusion, overexpression of cyclin D2 mRNA was found to be an almost constant feature in B-CLL and LPL. Therefore, it led us to hypothesize, with the support of data from some transfection experiments previously reported in murine hematopoietic cell lines, that cyclin D2 might play a role in B-CLL pathogenesis, possibly by preventing cells from programmed cell death.     

Rapamycin-induced G1 arrest in cycling B-CLL cells is associated with reduced expression of cyclin D3, cyclin E,cyclin A,and survivin

In B-cell chronic lymphocytic leukemia (B-CLL), malignant cells seem to be arrested in the G(0)/early G(1) phase of the cell cycle, and defective apoptosis might be involved in disease progression. However, increasing evidence exists that B-CLL is more than a disease consisting of slowly accumulating resting B cells: a proliferating pool of cells has been described in lymph nodes and bone marrow and might feed the accumulating pool in the blood. Rapamycin has been reported to inhibit cell cycle progression in a variety of cell types, including human B cells, and has shown activity against a broad range of human tumor cell lines. Therefore, we investigated the ability of rapamycin to block cell cycle progression in proliferating B-CLL cells. We have recently demonstrated that stimulation with CpG-oligonucleotides and interleukin-2 provides a valuable model for studying cell cycle regulation in malignant B cells. In our present study, we demonstrated that rapamycin induced cell cycle arrest in proliferating B-CLL cells and inhibited phosphorylation of p70s6 kinase (p70(s6k)). In contrast to previous reports on nonmalignant B cells, the expression of the cell cycle inhibitor p27 was not changed in rapamycin-treated leukemic cells. Treatment with rapamycin prevented retinoblastoma protein (RB) phosphorylation in B-CLL cells without affecting the expression of cyclin D2, but cyclin D3 was no longer detectable in rapamycin-treated B-CLL cells. In addition, rapamycin treatment inhibited cyclin-dependent kinase 2 activity by preventing up-regulation of cyclin E and cyclin A. Interestingly, survivin, which is expressed in the proliferation centers of B-CLL patients in vivo, is not up-regulated in rapamycin-treated cells. Therefore, rapamycin interferes with the expression of many critical molecules for cell cycle regulation in cycling B-CLL cells. We conclude from our study that rapamycin might be an attractive substance for therapy for B-CLL patients by inducing a G(1) arrest in proliferating tumor cells.     

Expression of myelomonocytic antigens on chronic lymphocytic leukemia B cells correlates with their ability to produce interleukin 1

We analyzed the expression of myelomonocytic-associated antigens on lymphocytes from B cell chronic lymphocytic leukemia (B-CLL) patients. Blood mononuclear cells were depleted of monocytes by one-step Percoll density gradient centrifugation and tested for antigen expression by fluorescent microscopy and flow cytometry. The reactivity of patient lymphocytes was as follows: 26 of 31 were positive for CD14 (Myr), 22 of 31 for a monocyte Fc receptor (MFC-1), 22 of 31 for CD11b (C3bi receptor), eight of 31 for CD15 (Leu-M1), five of 18 for CD13 (My 7), seven of 18 for My 9, and five of 30 for Mo 2. The B lymphocytes of B- CLL patients were also tested for the ability to produce interleukin 1 (IL-1) after depletion of monocytes and T lymphocytes. In 13 of 17 cases, B lymphocytes of patients produced IL-1 as detected in a mouse thymocyte proliferation assay and, in selected cases, a radioimmunoassay specific for IL-1 beta. The 13 cases that produced IL- 1 were also positive for the expression of one or more myelomonocytic- associated antigens, whereas the four cases that did not produce IL-1 lacked expression of these antigens. In conclusion, the malignant B cells of B-CLL patients frequently express a variety of antigens generally considered specific for myelomonocytic cells, and expression of these antigens is associated with the ability to produce IL-1.     

B-cell chronic lymphocytic leukemia cells express a surface membrane phenotype of activated,antigen-experienced B lymphocytes

B-cell chronic lymphocytic leukemia (B-CLL) is considered an accumulative disease of antigen-naive CD5(+) B lymphocytes that circulate in the resting state. However, to evaluate the possibility that B-CLL cells resemble antigen-experienced and activated B cells, we analyzed the expression of markers of cellular activation and differentiation on CD5(+)CD19(+) cells from B-CLL patients and from age-matched healthy donors. The leukemic cells from all B-CLL patients, including those that lack significant numbers of V gene mutations, bear the phenotype of activated B cells based on the overexpression of the activation markers CD23, CD25, CD69, and CD71 and the underexpression of CD22, Fcgamma receptor IIb, CD79b, and immunoglobulin D that are down-regulated by cell triggering and activation. Furthermore, these leukemic cells resemble antigen-experienced lymphocytes in the underexpression of molecules that are down-regulated by cell triggering and in the uniform expression of CD27, an identifier of memory B cells. A comparison of the phenotypes of B-CLL patients with and without immunoglobulin V gene mutations suggests that the 2 subgroups differ both in specific marker expression (CD69, CD71, CD62 L, CD40, CD39, and HLA-DR) and in the time since antigenic stimulation, based on the reciprocal relationship of CD69 and CD71 expression. These findings imply that the leukemic cells from all B-CLL cases (irrespective of V gene mutations) exhibit features of activated and of antigen-experienced B lymphocytes and that the B-CLL cells that differ in immunoglobulin V genotype may have different antigen-encounter histories.     

Four ricin chain A-based immunotoxins directed against the common chronic lymphocytic leukemia antigen: in vitro characterization

The common B-chronic lymphocytic leukemia (B-CLL) antigen (cCLLa) appears to be ideal for targeted immunotherapy in that it is the most prevalent and disease-restricted marker in B-CLL. To assess this potential, we developed four immunotoxins (ITs) of anti-cCLLa monoclonal antibody CLL2m (an IgG2a kappa), using ricin chain A (RTA) or its deglycosylated derivative (dgA), each conjugated to either the whole IgG molecule or its Fab' fragment. Each IT was tested in vitro for specificity and cytotoxic activity (assessed by protein synthesis inhibition [PSI] and by cell kill [CK] in the clonogenic assay) against B-CLL cells. RTA-based anti-CD5 ITs and enriched normal B and T lymphocytes were used as controls. Each IT exhibited antigen-specific, dose-dependent activity. Thus, whereas B-CLL cells exhibited dose- dependent PSI and CK (whether the B-CLL clone was CD5+ or CD5-), normal B (cCLLa-/CD5-) and T lymphocytes (cCLLa-/CD5+) remained unaffected. IT potency was independent of toxin glycosylation, but was slightly influenced by antibody valence; divalent ITs were twice as potent as monovalent ITs (IC50, 2.3 v 7.1 x 10(-11) mol/L; CK, 2.6- v 2.0-log reached with 524 v 1,072 IT molecules bound/cell, respectively). In the presence of ammonium chloride or Verapamil, IT-induced CK was enhanced 10- to 80-fold. These data suggest that the cCLLa is a promising target for IT-based immunotherapy of B-CLL in vivo and ex vivo.     

Cell cycle regulatory proteins and apoptosis in B-cell chronic lymphocytic leukemia.

BACKGROUND AND OBJECTIVES: The pathogeny of B-cell chronic lymphocytic leukemia (B-CLL) involves both deregulated proliferation and inhibition of cell death. A particular role in the regulation of these phenomena is played by proteins involved in early G1 phase regulation: pRb kinases: cyclin-dependent kinases (cdk): cdk4 and cdk6 activated by cyclins D, and universal cdk inhibitor p27(Kip1). DESIGN AND METHODS: We determined by flow cytometry the expression of p27(Kip1) and cyclins D (D2 and D3) in populations of peripheral blood lymphocytes obtained from 59 (for p27(Kip1)) and 31 (for cyclins D) previously untreated patients with B-CLL, and compared them with cell cycle parameters, cell viability and apoptosis in 72-hour cultures in medium only. As a control we determined the expression of p27(Kip1), cyclin D2 and D3 in peripheral blood CD5+/CD19+ lymphocytes from 15 healthy donors. RESULTS: p27(Kip1) was present in nearly 100% of lymphocytes in all B-CLL populations tested. Its cellular content estimated semiquantitatively by specific mean fluorescence intensity was higher than in normal CD5+/CD19+ lymphocytes, p27(Kip1) was inversely correlated with patients' age and not correlated with other clinical variables, cell cycle or apoptosis rate. Cyclin D2 was detectable in 25 out of 31, and cyclin D3 in all B-CLL lymphocytes populations studied. In contrast to p27Kip1 present in all CD5+/CD19+ lymphocytes, both cyclins were detected only in a subset of neoplastic cells: 27.5 to 87% (mean 51.2) for cyclin D2 and 20.3 to 98% (mean 76.5) for cyclin D3. In cyclin D2- and D3-positive normal CD5+/CD19+ lymphocytes and B-CLL cell populations, cyclin D3 was expressed in a higher percentage of cells than cyclin D2. Both cyclin D2-and cyclin D3-positive fractions of B-CLL cells were, on average, larger than corresponding fractions of normal CD5+/CD19+ peripheral blood lymphocytes. INTERPRETATION AND CONCLUSIONS: Our results indicate that cyclin D3 plays an important role in the regulation of normal and neoplastic CD5+/CD19+ cells, and point to the possibility of the exit of a number of CLL lymphocytes from quiescence.     

Transendothelial migration of lymphocytes in chronic lymphocytic leukaemia is impaired and involved down-regulation of both L-selectin and CD23

Chronic lymphocytic leukaemia (B-CLL) is characterized by a progressive accumulation of B lymphocytes in blood and bone marrow and high concentrations of soluble CD23 and L-selectin are found in the serum of these patients. In this study lymphocytes from normal subjects and patients with B-CLL were allowed to undergo transendothelial migration across confluent layers of human umbilical vein endothelial cells. Lymphocytes in B-CLL samples showed an impaired capacity to migrate while the minor proportion of normal T cells was enriched by a mean of 2.5-fold in the transmigrated lymphocytes. In contrast, the ratio of B to T lymphocytes in normal preparations was unchanged in the transmigrated population. The expression of adhesion molecules on B-CLL lymphocytes before and after transendothelial migration was studied by flow cytometry which showed that 71 +/- 5% of L-selectin was lost from the surface of transmigrated lymphocytes. T and B cells from normal subjects also showed a major loss of L-selectin after transmigration. B-CLL lymphocytes and normal B cells expressed CD23 but this molecule was down-regulated following transendothelial migration, whereas the expression of VLA-4, ICAM-1, LFA-1 and CD44 was unchanged. Lymphocytes incubated with oxidized ATP, an irreversible inhibitor of P2Z/P2X7 purinoceptors, retained their capacity for transendothelial migration and showed the same loss of L-selectin as control leukaemic lymphocytes. Our results show that B-CLL lymphocytes have impaired ability for transendothelial migration compared to normal peripheral blood lymphocytes. Moreover, transendothelial migration involves a universal loss of L-selectin and CD23 from lymphocytes which suggests that the high serum levels of soluble L-selectin and CD23 observed in B-CLL may be generated by shedding during the process of transendothelial migration.     

Sensitization of B-cell chronic lymphocytic leukemia cells to recombinant immunotoxin by immunostimulatory phosphorothioate oligodeoxynucleotides

A recombinant anti-CD25 immunotoxin, LMB-2, has shown clinical efficacy in hairy cell leukemia and T-cell neoplasms. Its activity in B-cell chronic lymphocytic leukemia (B-CLL) is inferior but might be improved if B-CLL cells expressed higher numbers of CD25 binding sites. It was recently reported that DSP30, a phosphorothioate CpG-oligodeoxynucleotide (CpG-ODN) induces immunogenicity of B-CLL cells by up-regulation of CD25 and other antigens. The present study investigated the antitumor activity of LMB-2 in the presence of DSP30. To this end, B-CLL cells from peripheral blood of patients were isolated immunomagnetically to more than 98% purity. Incubation with DSP30 for 48 hours augmented CD25 expression in 14 of 15 B-CLL samples, as assessed by flow cytometry. DSP30 increased LMB-2 cytotoxicity dose dependently whereas a control ODN with no CpG motif did not. LMB-2 displayed no antitumor cell activity in the absence of CpG-ODN as determined colorimetrically with an (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. In contrast, B-CLL growth was inhibited in 12 of 13 samples with 50% inhibition concentrations (IC(50)) in the range of LMB-2 plasma levels achieved in clinical studies. Two samples were not evaluable because of spontaneous B-CLL cell death in the presence of DSP30. Control experiments with an immunotoxin that does not recognize hematopoietic cells, and an anti-CD22 immunotoxin, confirmed that sensitization to LMB-2 was specifically due to up-regulation of CD25. LMB-2 was much less toxic to normal B and T lymphocytes compared with B-CLL cells. In summary, immunostimulatory CpG-ODNs efficiently sensitize B-CLL cells to a recombinant immunotoxin by modulation of its target. This new treatment strategy deserves further attention.     

Morphological variants of leukemic cells in B chronic lymphocytic leukemia are associated with different T cell and NK cell abnormalities

B chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease. The different morphological variants of leukemic B cells appear to define different clinical groups of patients. Several abnormalities have been found in T lymphocytes and natural killer (NK) cells from B-CLL patients. We have investigated the phenotypic and functional characteristics of purified CD2+ cells from B-CLL patients at Binet's stage A and classified according to the neoplastic B lymphocyte morphology criteria: 32 patients with typical B-CLL and 12 patients with atypical B-CLL. Forty-three age and sex matched healthy controls were also studied. In fresh purified CD2+ cells from typical B-CLL patients, percentages of CD4+, CD4+CD45RA+, CD8+CD45RA+ T lymphocytes and CD3−CD56+ (NK) cells were significantly higher than those found in atypical B-CLL patients. However, in DC2+ cells from typical B-CLL patients, percentages of CD3+, CD3+DR+, CD8+, CD4+CD45RO+, and CD3+CD56+ cells were significantly lower than those found in atypical B-CLL patients. Increased percentage of NK cells was only found in typical B-CLL patients. The proliferative response and the production of interleukin (IL)-2 and IL-4 by phytohemagglutinin (PHA) stimulated CD2+ cells were significantly higher in typical B-CLL patients than in atypical B-CLL patients. We concluded that different patterns of phenotypic and functional alterations in the T lymphocytes and NK cells of B-CLL patients are found in patients with typical or atypical B-CLL defined according to the morphology of the leukemic cells. Am. J. Hematol. 55:175–182, 1997. © 1997 Wiley-Liss, Inc.     

Glutathione depletion in chronic lymphocytic leukemia B lymphocytes.

Glutathione (GSH) content may be the major determinant of a cell's sensitivity to cytotoxic alkylating agents. In the present study, the GSH concentration was determined in lymphocytes isolated from the blood of normal subjects and patients with chronic lymphocytic leukemia (CLL). Comparable levels were found in both types of cells. Incubation for 20 hours led to a decrease in GSH to 51% of baseline values in CLL B cells. Under the same conditions, normal B- or T-lymphocyte GSH content remained constant. GSH depletion was shown to be a characteristic of the B-CLL B lymphocyte. It was not found in the T cells of patients with B-CLL or in cells from patients with T-CLL. Chlorambucil (CLB) contributes to the decrease in GSH in B-CLL lymphocytes; after incubation with the drug, lower levels of GSH were found than in the normal B or T lymphocytes, B-CLL T cells, or T-CLL (CD4 or CD8) cells. GSH depletion of CLL B lymphocytes may be related to the greater therapeutic efficacy of CLB in B-CLL than in T-CLL.     

The role of CD45 in the activation, proliferation and differentiation of human B lymphocytes.

We examined the role of CD45 antigen in human B cell function, using the anti-CD45 antibody, T29/33. The addition of T29/33 to B cells inhibited the proliferative response induced by various polyclonal B cell activators in a dose-dependent manner at concentrations of 0.01 to 10 micrograms/ml. Kinetic analysis indicated that T29/33 exerted its inhibitory effect when added within the first 24 h of culture initiation during a 72-h culture period, but had little effect when added at 48 h. Pre-treatment of high-density B cells with T29/33 antibody for 48 h showed a marked inhibitory effect on the proliferative response of these B cells when they were subsequently stimulated with SAC. Antibody to CD45 appeared to block the G0(G1) to S phase transition in the cell cycle analysis by propidium iodide staining. T29/33 antibody suppressed the RNA and DNA synthesis induced by SAC stimulation in B cells. These data suggested that small resting B cells were sensitive to anti-CD45-induced suppression. T29/33 antibody also suppressed immunoglobulin synthesis in B cells, independently of its suppressive effect on proliferation. These results indicated that CD45 antigen defined by T29/33 is involved in the activation, proliferation, and differentiation signals of human B lymphocytes.     

Analysis of signal transduction in B chronic lymphocytic leukemia cells

We report here experiments on the analysis of cellular signal transduction in a series of patients with chronic B cell disorders (B cell chronic lymphocytic leukemia [B-CLL] and prolymphocytic leukemia). We compared the response of the leukemic cells with primary external signals (interleukin 2 [IL-2] or B cell differentiation factors [BCDF or IL-6]) with their response to secondary inducers (the phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA] or the calcium ionophore A23187) that circumvent the first part of the signal transduction pathway by directly activating the key enzyme protein kinase C. One BCDF was synthesized by mitogen-activated peripheral blood B lymphocytes; a second BCDF was constitutively produced by the human bladder carcinoma cell line T24. Changes in morphology, Tac (IL-2 receptor) expression, RNA synthesis measured by 3H-uridine uptake, and immunoglobulin production tested by enzyme-linked immunosorbent assay were used as parameters of successful signal transduction. TPA alone and TPA plus A23187 (synergistically) effectively initiated differentiation in all the leukemia cases. Neither IL-2 nor BCDF (singly or in combinations) caused equivalent responses. On the other hand, IL-2 and BCDF produced a substantial differentiation effect on normal B lymphocytes. Our data suggest that (a) B-CLL cells are able to respond to direct stimulation of the second messenger pathway (through protein kinase C) but not to the physiological stimuli IL-2 or BCDF; (b) the defect in signal transduction appears to be located upstream of protein kinase C (a possible candidate is a G protein); (c) malignant B cells may spontaneously or after treatment with inducers express the IL- 2 receptor (Tac antigen) in the absence of a functional differentiating response to IL-2; and (d) signs of proliferation/differentiation in B- CLL samples after incubation with IL-2 or BCDF might be due to contamination of the cell populations with residual normal B cells.     

Aberrant CD8 expression in B-chronic lymphocytic leukemia: report of five cases

The expression of T cell-associated antigens on B cell non-Hodgkin lymphoma is rare. We describe 5 cases of B-chronic lymphocytic leukemia (B-CLL) with aberrant expression of CD8 on B cells. These B-CLL presented a typical immunophenotype CD19+, CD23+, FMC7- and CD5+ (except for 1 case) with a monotypic expression of surface immunoglobulin light chain kappa. The CD8 expression was confirmed on B-CLL cells by two-color flow cytometry staining. We could not find coexpression of CD3 or CD4 on B-CLL cells. The clinical implications, the sensitivity to therapy and the prognostic outcome of this aberrant expression remains to be determined.