B cells in chronic lymphocytic leukaemia. Comparative analysis of blood and bone marrow

A study was performed on cell suspension from peripheral blood and bone marrow aspirates and on cryostat sections from bone marrow biopsies in order to investigate the membrane phenotype of neoplastic B cells in chronic lymphocytic leukaemia (B-CLL). The immunological analyses, performed on 43 patients, included rosetting ability with sheep and mouse erythrocytes, evaluation of surface immunoglobulins and reactivity with anti-HLA-DR, UCHT 1 (OKT-3 like) and RFA-1 (OKT-1 like) monoclonal antibodies. The results demonstrate that neoplastic B lymphocytes in B-CLL display an identical phenotype in peripheral blood and bone marrow. Possible interpretations on the origin of proliferating cells in B-CLL are discussed.     

In vitro maturation of B cells in chronic lymphocytic leukemia. I. Synergistic action of phorbol ester and interleukin 2 in the induction of Tac antigen expression and interleukin 2 responsiveness in leukemic B cells

Recent evidence indicates that interleukin 2 (IL 2), formerly thought to serve as growth factor exclusively for activated T cells, is directly involved in human B cell differentiation. We have investigated the role of IL 2 and IL 2 receptors (as defined by monoclonal anti-Tac antibody) in the phorbol ester-induced in vitro maturation of leukemic B cells from patients with chronic lymphocytic leukemia (CLL). Peripheral blood lymphocytes from B cells from CLL patients with high (greater than 10(5)/microliters) white blood cell counts were depleted of residual T lymphocytes and low-density cells (primarily macrophages) by consecutive steps of E rosetting, complement-mediated lysis of OKT3+ and OKT4+ cells, and Percoll density gradient centrifugation. No OKT3+ T cells were detectable in these cell populations before or after culture. When incubated for 3 days with phorbol ester plus recombinant human IL 2 (rIL 2), 12 to 57% of highly purified B cells from four of five tested patients expressed Tac antigen. Both phorbol ester and rIL 2 were required for maximal Tac antigen expression. Functional studies revealed that phorbol ester-activated (but not resting) CLL B cells responded to rIL 2 with [3H]thymidine incorporation and with enhanced secretion of IgM. Tac+ B cells were isolated in two cases on a fluorescence-activated cell sorter. In one patient, stimulation of Tac+ B cells with rIL 2 resulted in enhanced [3H]thymidine incorporation but no change in IgM secretion, as compared with Tac- B cells; in the second patient, stimulation of Tac+ B cells with rIL 2 did not result in [3H]thymidine uptake, but did result in significant IgM secretion. These findings indicate that certain leukemic B lymphocytes can be induced to express IL 2 receptors and respond to IL 2. The use of resting clonal B cell populations arrested at distinct stages of differentiation may help to better define the stage(s) at which IL 2 acts directly on B cells to induce proliferation and/or terminal differentiation.     

Comparative analysis of membrane glycoproteins of normal and chronic lymphatic leukemia B lymphocytes by lectins

Eight plant lectins were used to investigate membrane alterations in lymphocytes from patients with chronic lymphocytic leukemia (CLL). By rosetting with lectins attached to latex particles, the cell percentages with the abundance of each lectin receptor were compared in B normal and leukemic lymphocytes. Comparing these data with the number of lectin molecules bound to each cell and the affinity, which are values calculated with 125I-labeled lectins, it was possible to deduce differences in the composition of glycoproteins in B normal and B-CLL lymphocytes membrane. Compared to B normal, B-CLL lymphocytes had fewer receptors for WGA and more for Lens culinaris, SBA and Tetragonolobus purpureus lectins. Receptors for Concanavalin A, Pisum sativum, PHA and Tetragonolobus purpureus showed a higher affinity with B normal lymphocytes, while the other lectins assayed showed more affinity with B-CLL lymphocytes. So, it is possible to establish a comparative analysis about the plasma membrane glycoproteins in the B normal and CLL lymphocytes by lectin binding studies.     

Aberrant expression of TRAIL in B chronic lymphocytic leukemia (B-CLL) cells

Analysis of peripheral blood (>85% CD19+/CD5+ B) lymphocytes, obtained from 44 patients affected by B chronic lymphoid leukemia (B-CLL), showed that surface TNF-related apoptosis inducing ligand (TRAIL) was expressed in all samples and at higher levels with respect to unfractionated lymphocytes and purified CD19+ B cells, obtained from 15 normal blood donors. Of note, in a subset of B-CLL samples, the addition to B-CLL cultures of a TRAIL-R1-Fc chimera, which binds at high affinity to surface TRAIL, significantly decreased the percentage of viable cells with respect to untreated control B-CLL cells, suggesting that surface TRAIL may play an unexpected role in promoting B-CLL cell survival. In spite of the majority of B-CLL lymphocytes expressed variable surface levels of "death receptors" TRAIL-R1 and TRAIL-R2, the addition in culture of recombinant TRAIL increased (>20% vs. controls) the degree of spontaneous apoptosis in only 11/44 of the B-CLL samples, had no effect in 19/44, while it significantly increased leukemic cell survival in 14/44. Taken together, these findings suggest that an aberrant expression of TRAIL might contribute to the pathogenesis of B-CLL by promoting the survival in a subset of B-CLL cells.     

Cortisol is associated with low frequency of interleukin 10–producing B cells in patients with atherosclerosis

It is accepted that inflammation plays a critical role in the development of atherosclerosis; the pathogenesis is not clear. B‐cell–produced interleukin (IL) 10 is an immune regulatory cytokine that can inhibit immune inflammation. This study tests a hypothesis that a psychological stress hormone, cortisol, suppresses IL‐10 expression in peripheral B cells of patients with atherosclerosis. Peripheral blood samples were collected from patients with coronary artery atherosclerosis. B cells were isolated from the blood samples to be analyzed for the expression of IL‐10 and micro RNA (miR) 98 by real‐time polymerase chain reaction. We observed that the frequency of IL‐10⁺ B cell was less in patients with atherosclerosis than healthy controls. The serum cortisol levels were higher in the patients than that in healthy controls. Peripheral B‐cell frequency was negatively correlated with the serum cortisol levels. Exposure of B cells to cortisol increased the expression of miR‐98 in B cells. Cortisol also inhibited the expression of IL‐10 in B cells, in which miR‐98 played a critical role. Treating B cells from atherosclerosis patients with anti–miR‐98 liposomes reversed the ability of expression of IL‐10 in the cells. The expression of IL‐10 is suppressed in peripheral B cells, which can be up regulated by anti–miR‐98 liposomes.     

Natural anti-bacterial activity against Salmonella typhi by human T4+ lymphocytes armed with IgA antibodies

Peripheral blood mononuclear cells from normal volunteers possess natural anti-bacterial (NA) activity against S. typhi that can be measured in a 2-hr in vitro assay. Employing fractionation on nylon wool columns, Percoll gradients, plastic adherence, and E rosetting, the effector cell of NA activity appeared to be a lymphocyte of the T lineage rather than a macrophage, a B lymphocyte, or a large granular cell. Moreover, complement-dependent killing with monoclonal antibodies such as OKM1, OKB7, OKT8, 5.9 and the anti-natural killer cells AB8.28 did not reduce NA activity. On the contrary, this was completely inhibited when OKT3, OKT11, or OKT4 antibodies and complement were used to pretreat the effector lymphocytes. Indeed, T4+ cells sorted with a FACS displayed an extremely high NA activity against S. typhi. By pretreatment of peripheral lymphocytes with F(ab')2 fragments against human IgA, the NA activity was blocked. It is therefore suggested that NA activity by human cells might be a mechanism of defense against infections, acting as antibody-dependent cellular cytotoxicity expressed by T4+ lymphocytes coated with preexisting anti-Salmonella IgA antibodies.     

Induction of proliferation and Ig production in human B leukemic cells by anti-immunoglobulins and T cell factors

The proliferation and differentiation of human leukemic B cells (B-CLL cells) with anti-Ig and T cell-derived helper factors are described. Stimulation of B-CLL cells with anti-Ig and T helper factors could induce proliferation as well as differentiation into IgM- and IgG-producing cells. Neither anti-Ig nor T helper factors alone could induce any proliferation and/or differentiation of B-CLL cells. Not only whole molecules of anti-Ig but also F(ab')2 fragments could induce proliferation and differentiation of B-CLL cells in the presence of T helper factors, but monovalent Fab' fragments were not effective. Induction of both IgM and IgG with the same idiotype was confirmed by immunofluorescent and SDS-PAGE analysis. By employing an IL 2-dependent cytotoxic T cell line and a TRF-responsive B cell line, T cell factors were separated into a fraction with IL2 activity but no TRF activity and a fraction with TRF activity but no IL 2 activity by chromatofocusing. Anti-Ig and IL 2 fraction could induce proliferation of B-CLL cells, but TRF fraction was not effective for the induction of proliferation in anti-IG-stimulated cells. For IgM and IgG production, anti-Ig and both IL 2 and TRF fractions were required. Depletion of IL 2 fraction in the first 2 days' culture inhibited Ig production, whereas the absence of TRF fraction in the first 2 days did not show any inhibitory effect on Ig production.     

A human B lymphocyte antigen (P-76) shared by B-cell chronic lymphocytic leukemia cells and hairy cell leukemia cells

A membrane antigen with an apparent specificity to B lymphocytes was detected with immunochemical techniques and its properties were analyzed. Anti-B-CLL serum was raised in a rabbit by immunization with B-cell chronic lymphocytic leukemia (B-CLL) cells. This anti-B-CLL serum was absorbed with erythrocytes, liver homogenate and insolubilized immunoglobulins. After further absorption with T-CLL cells, chronic myelocytic leukemia (CML) cells and acute myelocytic leukemia (AML) cells, the anti-B-CLL serum still reacted with peripheral blood B lymphocytes, B-CLL cells and hairy cell leukemia (HCL) cells. In contrast, no reactivity was seen with peripheral blood T lymphocyte or monocytes, or leukemia cells of non-B cell origin. An immunoprecipitation of radiolabeled cell surface proteins was attempted using the anti-B-CLL serum in the presence of Staphylococcus Aureus Cowan 1 (SaCl), and the precipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A membrane antigen with an apparent molecular weight of 76,000 daltons (P-76) was immunoprecipitated with the anti-B-CLL serum from the lysates of normal B lymphocyte, B-CLL cells and HCL cells. The antigen (P-76) is not composed of disulfide-linked subunits and has no structural relationship with HLA-DR (Ia-like) antigens or other known antigens. These results suggest that this antigen is B-lymphocyte specific, and favour the B-lymphocyte nature of HCL cells.     

Synergistic effect of recombinant IL 2 and interferon-gamma on the proliferation of human monoclonal lymphocytes

We studied the effect of interferon-gamma (IFN-gamma) on the proliferation of lymphocytes from 10 B-type chronic lymphocytic leukemia (B-CLL) patients. In no instance did IFN-gamma induce a proliferative response whether used alone or in combination with anti-mu antibody (Ab). This was observed regardless of the responsiveness of a given patient's cells to interleukin 2 (IL 2) and to B cell growth factor (BCGF). In contrast IFN-gamma strongly and reproducibly synergized with IL 2 (but not with BCGF) to support B-CLL proliferation in five of the 10 patients. The effect of IFN-gamma was dose related and could be inhibited by an anti-IFN-gamma monoclonal Ab. A monoclonal Ab toward the IL 2 receptor molecule was also inhibitory. Preincubation with IFN-gamma potentiated the responsiveness of B-CLL to IL 2 in secondary cultures, showing that IFN-gamma exerts its effect before that of IL 2.     

Dual fluorochrome analysis of human B lymphocytes: phenotypic examination of resting, anti-immunoglobulin stimulated, and in vivo activated B cells

B cell-enriched preparations were prepared from human peripheral blood and lymphoid tissues by the depletion of T cells and monocytes. Only B cells by virtue of their staining with anti-B1 conjugated to fluorescein were additionally examined. Dual fluorescence staining and flow cytometric analysis demonstrated that the majority of "resting" human peripheral blood and splenic B cells co-express the B cell-restricted B1 and B2 antigens and lack B5, a B cell-restricted activation antigen, and interleukin 2 receptor (IL 2R). In contrast, nearly 2/3 and 1/3 of B1+ cells isolated from lymph node expressed IL 2R and B5 antigens, respectively. When B1+ B cells from peripheral blood and spleen were "activated" by anti-Ig, they lost the B2 antigen and acquired the B5 and/or IL 2R antigens. 2/3 of B1+ cells strongly expressed IL 2R, and up to 1/2 of B1+ cells co-expressed B5. Delineation of increased numbers of B1+ cells that co-express B5 and/or IL 2R within lymphoid tissues obtained from patients with diseases characterized by "activated" B cells provides in vivo confirmation that these phenotypic changes correlate with B cell activation. We believe that the identification and isolation of these and similar subsets of cells defined by differing cell surface phenotypes should further our understanding both of normal B cell activation and the pathophysiology of B cell disease states.     

Comparison of different markers on blood lymphocytes of chronic lymphocytic leukemia

Mononuclear cells (MNC) of 17 patients suffering from B chronic lymphocytic leukemia (B-CLL) were analysed by various immunological methods. The B cell nature of CLL cell was determined by classical tests (MRBC-rosette-test, immunofluorescence test for detection of membrane bound immunoglobulins). The cytochemical detection of the new T-cell marker dipeptidyl peptidase IV (DP IV) was found to be suitable for the characterization of B-CLL. The B-CLL cells showed granular pattern of alpha-naphthylacetate esterase (ANAE) reaction and binding of the monoclonal pan T antibody BL-T2. These non typical reactions for normal B lymphocytes can be used for differential diagnosis of B-CLL in combination with other reliable T cell markers. Avoiding the separation of T cells, the mixed rosette assay was used to enumerate Fc-IgG-receptor bearing T(TG) and non T cells. Both cell populations were found to be significantly elevated in MNC of B-CLL.     

Generation of lymphokine-activated killer cell activity by low-dose recombinant interleukin-2 and tumor cells

The generation of lymphokine-activated killer (LAK) cells in vitro has been reported to require 100-1000 units of recombinant interleukin-2 (IL2). In this study we investigated the generation of human LAK cells with low-dose IL2 (1-10 U) in combination with human tumor cell lines. A significant LAK activity was generated within 3- to 5-days culture of PBL. Among six human tumor cell lines tested, the K562 cell line had the greatest stimulating activity, and the degree of cytotoxicity was comparative to that of PBL stimulated with higher doses of IL2 alone. The origin of this LAK activity was primarily the E(-) rosetting cell population. Cocultures of E- cells with 1 U/ml IL2 plus K562 had significantly higher cytotoxicity (P less than 0.05) compared to using E+ cells. Phenotypic analysis indicated that 1 U/ml IL2 plus K562 cell stimulation enhanced CD56+ and CD16+ cells. These studies suggest that very low dosages of IL2 with stimulator tumor cells can generate LAK activity comparable to that generated with high dosages of IL2 alone.     

Analysis of lymphocytes infiltrating the thyroid gland of Obese strain chickens

Lymphocytes were isolated from the infiltrated thyroid glands of 2- to 5-wk-old Obese strain (OS) chickens with spontaneous autoimmune thyroiditis (SAT). Immunofluorescence analysis performed by using a panel of monoclonal and polyclonal antibodies revealed that 60% of thyroid infiltrating leukocytes (TIL) were mature T cells, a large portion of which seemed to be in an activated state bearing Ia-like antigens (10%) as well as a surface determinant associated with T cell activation (16%), i.e., possibly the receptor for interleukin 2 (IL 2). Furthermore, a relatively high plasma cell content (5%) was observed. TIL exhibited high proliferative responses to T cell mitogens (concanavalin A, phytohemagglutinin) and IL 2, but only weak responses to the B cell mitogen LPS from Salmonella typhimurium. When injected into newly hatched, MHC-identical, irradiated normal chickens, TIL induced both the production of autoantibodies and thyroid infiltration. Peripheral lymphocytes from spleen and blood and thymocytes from the same OS donors had no effect. Analysis of chemically (cyclophosphamide) bursectomized OS chickens suggested that an intact B cell system was not obligatory for the induction of SAT. TIL from these chickens consisted of 77% T cells and less than 1% B lymphocytes, yet were capable of inducing severe thyroid infiltration upon transfer into normal recipients. These findings emphasize the importance of the T cell system in the initiation of SAT.     

Activation of human B lymphocytes. XIV. Characterization of the precursor of the pokeweed mitogen-induced anti-sheep red blood cell plaque-forming cell.

The precursor of the pokeweed mitogen (PWM)-induced anti-sheep red blood cell (SRBC) plaque-forming cell (PFC) in human peripheral blood was characterized. By a variety of purification procedures, it was demonstrated to be a lymphocyte with surface characteristics of a B cell. Furthermore, it was demonstrated to bind to sheep erythrocytes (E) and thus segregated with the E-rosetting T cells when T cell enrichment was performed by differential fractionation of E-rosetting cells. This binding of the PFC precursor to E was blocked by pretreating the lymphocyte with anti-human Ig before E rosetting, indicating that the PFC precursor specifically bound to SRBC by a surface Ig molecule with binding specificity for sheep red blood cell determinants. Hence, the precursor of the PWM-triggered anti-SRBC PFC is a B lymphocyte with surface Ig expressing specificity for SRBC.     

Membrane markers of the lymphocytes of human peripheral blood: techniques of study and normal values in adults]

Peripheral blood lymphocytes from normal subjects were evaluated for their cell surface markers. Normal values were determined in females and males for T-cells (E rosettes and active E rosettes) and for B cells (surface immunoglobulins, EA rosettes and EAC rosettes). Means expressed in percentage and in absolute number per cubic millimeter +/- standard error in normal subjects males and females.     

Binding sites for measles virus antigens on human B and T lymphocytes

The present study examined the differences in the binding of measles virus antigens to human peripheral blood lymphocyte (PBL) subpopulations. PBL binding sites for measles antigens were detected by an assay involving the rosetting of PBL to measles-infected HeLa cells (HeLa-K11). Three approaches were employed to examine whether measles virus antigen binding sites were present on restricted subpopulations of PBL. First, no significant difference in the proportion of HeLa-K11 forming clusters was observed with unfractionated cells in comparison with enriched B- or T-lymphocyte suspensions. Second, the profile of lymphocyte surface markers before and after adherence of PBL suspensions to HeLa-K11 cells was measured. No difference in the proportion of PBL forming E-rosettes or lymphocytes with Fc-IgG receptors, surface immunoglobulin, or complement receptors was observed. Finally, the percentage of B (Raji, B-35M, Bristow-7B) and T (Molt-3) cell human lymphoid cells which adhered to HeLa-K11 versus noninfected HeLa cells was compared. In all cases, a highly significant adherence of the lymphoid cell suspensions to HeLa-K11 cells was observed in comparison with uninfected HeLa cells. This is the first direct demonstration of binding sites for measles virus antigens present on both human B and T lymphocytes.     

Complement (C3) receptor-bearing lymphocyte-mediated cytotoxicity and lymphotoxin responses

The question of nonthymus-derived lymphocyte-mediated cytotoxicity was investigated with T and B cell subpopulations separated from the blood of normal donors. Mononuclear cells, T cells (E-RFC), and cell preparations enriched for B cells (non-E-RFC) by depletion of E-RFC gave negligible cytotoxic responses when incubated with either human melanoma or lung fibroblast target cells. In contrast, EAC and ZC rosetting cells separated from this same B-rich population consistently gave cytotoxic responses which were not dependent on either antibody or phagocytic cells. The cytotoxic effector cells appeared to be nonthymus-derived lymphocytes as characterized by C3 receptor rosetting and presence of surface membrane immunoglobulin on the majority of cells. In addition, supernatants from EAC-RFC cultures contained lymphotoxin (LT) activities which were eightfold higher than those of control E-RFC cultures. These findings suggest the existence of a nonthymus-derived cell cytotoxic effector mechanism, induced by the binding of membrane C3 receptors, which is independent of antibody.     

Differential fluorochromasia of human lymphocytes as measured by flow cytometry.

Peripheral human lymphocytes reacted with fluorescein diacetate and analyzed by flow cytometry produced a bimodal fluorescence distribution that was shown to be attributable to the differential staining of T and B lymphocytes. Lymphocytes were fractionated into rosetting (T cell) and nonrosetting (B cell) populations. Both subfractions were reacted with fluorescein diacetate and analyzed by flow cytometry. The rosetting fraction was more fluorescent than the nonrosetting fraction, and the analysis of an appropriate mixture of the subfractionated populations produced a fluorescence distribution very similar to that obtained with unfractionated lymphocytes.     

Serum levels of soluble interleukin-2 receptor (sIL-2R) in B-chronic lymphocytic leukemia.

By using an enzyme-linked immunosorbent assay, the levels of the soluble form of the interleukin-2 receptor (sIL-2R) were evaluated in the peripheral blood of 20 patients with cell chronic lymphocytic leukemia in different stages of disease and in supernatants obtained from enriched B cell suspensions. In either all serum samples or in one out of three supernatants, elevated levels of sIL-2R were found. This could indicate that the B leukemic cells release sIL-2R which in turn, for its potential capacity of binding circulating IL-2, could contribute to the abnormal immunoregulation which characterizes B-CLL. This finding, which needs further investigation, could have prognostic significance.     

Induction of DNA fragmentation in chronic B-lymphocytic leukemia cells

Chronic lymphocytic leukemia of B cell type (B-CLL) is a neoplastic disorder characterized by the accumulation of small resting lymphocytes in the periphery. The phenotype of these cells suggests that they are "frozen" at an early stage of maturation. Glucocorticoid hormones are commonly used to treat patients with B-CLL, resulting in a reduction in the peripheral lymphocyte count by an undefined mechanism. Here we report that glucocorticoids stimulate DNA fragmentation characteristic of a suicide process known as apoptosis or programmed cell death (PCD) in suspensions of cells from patients with B-CLL. The effects can be mimicked by Ca2+ ionophore and involve a sustained increase in the cytosolic Ca2+ concentration. Specific antibodies binding to membrane-associated IgM on the leukemic cells can also induce PCD by a similar mechanism. Phorbol esters block DNA fragmentation and cell killing in response to all of the agents, suggesting that activation of protein kinase C desensitizes the cells to PCD. Targeting the B-CLL cells with antibodies that induce an unbalanced, sustained Ca2+ increase may therefore represent a rational strategy for the destruction of leukemic cells.