Altered membrane-associated functions in chronic lymphocytic leukemia cells.

Peripheral blood lymphocytes consisting mainly of neoplastic B cells from patients with chronic lymphocytic leukemia (CLL cells) showed a markedly reduced response to the human B-cell mitogens anti-beta2 microglobulin, Sepharose-bound protein A and Sepharose-bound anti-human immunoglobulin (anti F(ab')2) in all of nine patients studied. On the other hand, CLL cells from three out of eight patients tested responded well to the calcium ionophore A23187. Sepharose-bound protein A and anti-beta2 microglobulin also failed to induce increased uptake of 86Rubidium (potassium analogue) in CLL cells as compared to B-cell-enriched preparations of normal peripheral blood lymphocytes. The capacity of CLL cells to cap various surface markers including beta2 microglobulin was reduced. On the other hand, surface concentrations of beta2 microglobulin were not reduced as measured by fluorescein-labelled anti-beta2-microglobulin in single-cell cytofluorometry. It is concluded that various membrane-associated events elicited by ligand-receptor interactions are altered or blocked in CLL cells.     

Chromosome studies in patients with T-CLL chronic lymphocytic leukemia and expansions of granular lymphocytes

Chromosome analyses were carried out in eight patients with lymphoproliferative disorders of mature T and NK cells. Three cases were characterized by an abnormal expansion of granular lymphocytes (GL), one by a lymphoma of GL with leukemic spread, and four by an OKT4-T-CLL. In four patients cytogenetic studies were performed on bone marrow cells; in seven patients peripheral blood lymphocytes were examined by either direct preparations or PHA-stimulated cultures. Six patients displayed a normal karyotype. Two cases belonging to the OKT4-T-CLL group had a chromosome number ranging from 44 to 47, with multiple numerical and structural clonal anomalies. Clonal anomalies could be a feature of patients with the more aggressive clinical course.     

Immunoglobulin synthesis and secretion by leukemic B cells from patients with chronic lymphocytic leukemia

We investigated the ability of purified E-rosette negative largely leukemic B cells from 15 patients with B-cell chronic lymphocytic leukemia (CLL) to synthesize and secrete IgM, IgA and IgG spontaneously or in the presence of purified autologous or allogeneic T4 cells from normal donors, in PWM-induced differentiation system. We observed moderate but significant IgM synthesis and secretion (19.7 +/- 8.9 micrograms/dl, n = 5) by leukemic B cells alone in 5 of 15 patients examined. These IgM concentrations were significantly higher (p less than 0.005) than those produced by purified E-rosette negative cells from normal donors (4.3 +/- 4.5 micrograms/dl; n = 6) in the absence of T cells. Purified E-rosette negative leukemic B cells alone from patients with CLL did not produce IgA or IgG. Addition of purified autologous or allogeneic T4 cells from normal donors resulted in significant increase of IgM production by leukemic B cells from certain patients or initiated IgM secretion in others. However, these IgM levels (73.9 +/- 56.6 micrograms/dl) were significantly lower (p less than 0.003) to those produced by mixtures of T4 cells and B cells form normal donors (211.6 +/- 58.0 micrograms/dl, n = 6). Addition of purified autologous or allogeneic T4 cells from normal donors to purified largely leukemic B cells from patients with CLL resulted in production of very small amounts of IgA in 4 of 15 patients (10.6 +/- 6.3 micrograms/dl vs 154.7 +/- 35.8 micrograms/dl produced by T4 and B cells from normal donors; n = 6), but did not support IgG synthesis and secretion. Purified T4 cells from certain patients with CLL exhibit defective helper function to immunoglobulin production by E-rosette negative cells from normal donors.     

Pro-apoptotic properties of hyperforin in leukemic cells from patients with B-cell chronic lymphocytic leukemia.

The effects of the hyperforin (HF), a natural phloroglucinol purified from Hypericum perforatum, were investigated ex vivo on leukemic cells from patients with B-cell chronic lymphocytic leukemia (B-CLL). HF was found to promote apoptosis of B-CLL cells, as shown by time- and dose-dependent stimulation of phosphatidylserine externalization and DNA fragmentation, by disruption of the mitochondrial transmembrane potential, caspase-3 activation and cleavage of the caspase substrate PARP-1. Moreover, HF-induced downregulation of Bcl-2 and Mcl-1, two antiapoptotic proteins that control mitochondrial permeability. HF also downregulated two proteins which are overexpressed by B-CLL patients' cells, the cell cycle inhibitor p27kip1 through caspase-dependent cleavage into a p23 form, and the nitric oxid (NO) synthase of type 2 (inducible NO synthase). This latter was accompanied by reduction in the production of NO known to be antiapoptotic in B-CLL cells. Preventing effects of the general caspase inhibitor z-VAD-fmk indicated that HF-promoted apoptosis of B-CLL cells was mostly caspase dependent. Furthermore, normal B lymphocytes purified from healthy donors appeared less sensitive to HF-induced apoptosis than B-CLL cells. These results indicate that HF may be of interest in the development of new therapies for B-CLL based on the induction of apoptosis and combination with cell cycle-dependent antitumor drugs.     

Expression of leucocyte-common antigen and large sialoglycoprotein on leukemic cells in B-cell chronic lymphocytic leukemia and non-Hodgkin's lymphoma

Patterns of leucocyte-common antigen (L-CA) and large sialoglycoprotein (LSGP) expression on leukemic peripheral blood lymphocytes of 13 patients with chronic lymphocytic leukemia (CLL), 17 with non-Hodgkin's lymphoma (NHL) in leukemic phase and one with hairy cell leukemia (HCL) have been examined by means of surface labelling and electrophoresis in 5% polyacrylamide gels. The 13 CLL, 10 of the 11 diffuse NHL and the six nodular poorly differentiated lymphocytic lymphoma (PDLL) patients fell into three groups according to expression of 210, 198 and 185k forms of L-CA. Group 1 (210 less than 198 less than 185k L-CA) included eight CLL and one diffuse NHL; Group 2 (210 greater than or equal to 198 and 185k L-CA) included four CLL, three diffuse NHL and four nodular PDLL; Group 3 (mainly 210k L-CA) included one CLL, six diffuse NHL and two nodular PDLL. A patient with diffuse large cell lymphoma and the HCL patient both had patterns of multiple, diffuse, very high Mr labelled glycoproteins. LSGP on these cells varied from nil to very high and levels were not related to L-CA patterns. Lymph node cells from five patients were also studied and were found to express larger numbers of L-CA forms and less LSGP than corresponding peripheral blood lymphocytes. Possible relationships of L-CA forms and LSGP to lymphocyte function and disease patterns are discussed.     

Concomitant presentation of acute myeloid leukemia with T-cell large granular lymphocytic leukemia

T-cell large granular lymphocyte leukemia (T-LGL) also known as T-cell chronic lymphocytic leukemia is rare and comprises a small minority of all small lymphocytic leukemias. The concomitant presentation of T-LGL with acute myeloid leukemia (AML) has not been previously reported. We present an elderly gentleman with concomitant T-LGL and AML (non-M3) diagnosed by a combination of morphologic evaluation, immunophenotyping by flow cytometry, and T-cell gene rearrangement studies. The patient was managed with combination AML chemotherapy. He remains alive and well seven months after initial diagnosis. A brief review of literature is also presented.     

Regulation of in-vivo differentiation of myeloid leukemic cells by antigen-specific helper T lymphocytes

There are clones of myeloid leukemic cells that can be induced to differentiate in vitro and in vivo by normal macrophage and granulocyte differentiation-inducing protein MGI-2 (= DF). The differentiation of these myeloid leukemic cells in vivo is regulated by a cell mediated immune response which requires T lymphocytes. We now show that differentiation of myeloid cells in vivo can be induced by antigen-specific helper T lymphocytes and that this is associated with the ability of the helper T cells to produce myeloid cell differentiation-inducing protein MGI-2. Antigen specific helper T cells can accumulate at a site that contains the antigen. It is suggested that migration in response to antigen of helper T cells producing differentiation factors may play an important role in inducing in vivo differentiation of leukemic cells.     

Selective toxicity of ethacrynic acid towards lymphocytes of chronic lymphocytic leukemia in vitro.

We have used the MTT colorimetric assay to determine the sensitivity to ethacrynic acid of lymphocytes from normal donors and of peripheral blood cells from leukaemia patients. Whereas normal lymphocytes and cells from acute or chronic myeloid leukaemia showed similar sensitivities (median inhibitory dose, ID50 = 20-22 microM), lymphocytes from chronic lymphocytic leukaemia patients were much more sensitive (ID50 = 6 microM). This result was found irrespective of the assay duration.     

Oligosaccharide components of surface glycoproteins from leukemic lymphocytes of chronic lymphocytic leukemia and non-Hodgkin's lymphoma

Oligosaccharide components of surface glycoproteins of leukemic cells have been studied in a group of eight patients with chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL). Labelled surface glycoproteins were separated by electrophoresis and treated with mild alkaline borohydride, prior to exclusion chromatography on Sephadex G-50. Five peaks were detected, of which peaks I and II consisted largely of N-linked chains and peaks III and IV of O-linked chains. Peak V contained monosaccharides and non-carbohydrate material. On both peripheral blood and lymph node cells of the CLL and nodular poorly differentiated lymphocytic lymphoma (NPDLL) patients studied, glycosylation was altered on leukocyte-common antigen (L-CA) forms of different relative molecular mass (Mr). The ratio of peak II oligosaccharides to those of peaks III and IV in different forms of L-CA correlated closely with Mr of the L-CA. Glycosylation of L-CA on cells of two patients with diffuse large cell lymphoma differed from that of the CLL and NPDLL patients, but was also to some extent related to Mr. Most of the oligosaccharides on the large sialoglycoprotein were O-linked, only a minor amount of N-linked being detected. Two surface glycoproteins of Mr 87 and 83 k differed markedly in their content of O- and N-linked chains. The specific glycosylation patterns described may have a role in control of cell behaviour and disease patterns of leukemia and lymphoma.     

Characterization of regulatory T cells in patients with B-cell chronic lymphocytic leukemia

The status of the immune system of patients with B-cell chronic lymphocytic leukemia (B-CLL) is not yet sufficiently characterized. Clinically, B-CLL patients present immunodeficiency increasing along with disease progression and signs of autoimmunity. In the current study, we evaluated the expression of FOXP3 in CD4+CD25hi T regulatory lymphocytes (Treg) and their influence on immune response against tumor and viral antigens in the complex system of peripheral blood mononuclear cells. In 80 B-CLL patients, the frequency of Treg (CD4+CD25hi FOXP3+) cells was significantly higher in B-CLL patients when compared to healthy volunteers (HV) and increased with the progression of the disease (median: 8.24% in stage A, 11.24% in stage B and 12.57% in stage C according to the Binet classification). The frequency of Treg showed no correlation with prognostic markers such as ZAP-70, CD38 and HLA-G. Notably, Treg frequency correlated with serum levels of TNF (r(2)=0.45, p=0.001). T-cell immune responses against epitopes derived from the tumor-associated antigens survivin, fibromodulin and RHAMM as well as from the influenza matrix protein were evaluated. Functionally, higher frequencies of Treg correlated with decreased T-cell responses against viral and tumor antigens. In conclusion, we detected higher frequencies of Treg in B-CLL patients than in HV. Furthermore, Treg constitute the crucial mechanism of immunosuppression in B-CLL patients.     

Carcinogen-induced repair and binding in the DNA of chronic lymphocytic leukemic lymphocytes.

Unscheduled DNA synthesis (repair) of carcinogen induced damage of the DNA of lymphocytes from 16 normal and 16 chronic lymphocytic leukemic (CLL) subjects was determined quantitatively for a standard dose of 10 micronM N-acetoxy-2-acetylaminofluorene (NA-AAF). Essentially all the CLL cases (15 of 16) had lower NA-AAF induced repair synthesis values than the normal subjects. Concurrent measurements for the levels of 3H-labeled 7,12-dimethyl-benz(a)anthracene (DMBA) to the DNAs of the normal and CLL lymphocytes after 18 h of culturing in 5 micronM DMBA have shown that 14 of 16 CLL cases had reduced levels of DNA bound carcinogen when compared to the normal individuals. Together these results suggest that CLL lymphocytes have a reduced repair synthesis because there is disproportionately less initial carcinogen-induced damage, and thereby, less substrate stimulation of repair enzymatic activity.     

Isolated anemia in patients with large granular lymphocytic leukemia (LGLL)

Patients with large granular lymphocytic leukemia (LGLL) frequently present with neutropenia. When present, anemia is usually accompanied by neutropenia and/or thrombocytopenia and isolated anemia is uncommon. We evaluated a cohort of 244 LGLL patients spanning 15 years and herein report the clinicopathologic features of 34 (14%) with isolated anemia. The patients with isolated anemia showed a significantly male predominance (p = 0.001), a lower level of hemoglobulin (p < 0.0001) and higher MCV (p = 0.017) and were less likely to have rheumatoid arthritis (p = 0.023) compared to the remaining 210 patients. Of the 34 LGLL patients with isolated anemia, 13 (38%) presented with pure red cell aplasia (PRCA), markedly decreased reticulocyte count and erythroid precursors, and more transfusion-dependence when compared to non-PRCA patients. There was no other significant clinicopathologic difference between PRCA and non-PRCA patients. 32 patients were followed for a median duration of 51 months (6–199). 24 patients were treated (11/11 PRCA and 13/21 non-PRCA patients, p < 0.02). The overall response rate to first-line therapy was 83% [8/11 (72.7%) for PRCA, 12/13 (92.3%) for non-PRCA], including 14 showing complete response and 6 showing partial response with a median response duration of 48 months (12–129). Half of non-PRCA patients who were observed experienced progressive anemia. During follow-up, no patients developed neutropenia; however, 5/27 (18.5%) patients developed thrombocytopenia. No significant difference in overall survival was noted between PRCA and non-PRCA patients. In summary, this study demonstrates the unique features of LGLL with isolated anemia and underscores the importance of recognizing LGLL as a potential cause of isolated anemia, which may benefit from disease-specific treatment. LGLL patients with PRCA were more likely to require treatment but demonstrated similar clinicopathologic features, therapeutic responses, and overall survival compared to isolated anemia without PRCA, suggesting PRCA and non-PRCA of T-LGLL belong to a common disease spectrum.Subject terms: Leukaemia, Leukaemia     

Improving therapy of chronic lymphocytic leukemia with chimeric antigen receptor T cells

Adoptive cell immunotherapy for the treatment of chronic lymphocytic leukemia (CLL) has heralded a new era of synthetic biology. The infusion of genetically engineered, autologous chimeric antigen receptor (CAR) T cells directed against CD19 expressed by normal and malignant B cells represents a novel approach to cancer therapy. The results of recent clinical trials of CAR T cells in relapsed and refractory CLL have demonstrated long-term disease-free remissions, underscoring the power of harnessing and redirecting the immune system against cancer. This review will briefly summarize T-cell therapies in development for CLL disease. We discuss the role of T-cell function and phenotype, T-cell culture optimization, CAR design, and approaches to potentiate the survival and anti-tumor effects of infused lymphocytes. Future efforts will focus on improving the efficacy of CAR T cells for the treatment of CLL and incorporating adoptive cell immunotherapy into standard medical management of CLL.     

Phosphomonoester concentrations differ between chronic lymphocytic leukemia cells and normal human lymphocytes

Levels of phospholipid-related metabolites of chronic lymphocytic leukemia lymphocytes (CLL) and normal human lymphocytes were quantified using phosphorus magnetic resonance spectroscopy. The CLL cells versus normal lymphocytes showed significant increases of phosphoethanolamine(Etn-P) (8.11+/-2.10 mean+/-S.E., micromol/g wet weight, n=12 versus 3.63+/-1.10, n=3, P相似文献   

Establishment of a lymphoid cell line from leukemic cells of a patient with chronic lymphocytic leukemia

Two lymphoid cell lines were established from a patient with chronic lymphocytic leukemia by infecting blood cells with Epstein-Barr virus (EBV). Studies of morphology, glucose-6-phosphate dehydrogenase, malic enzyme, immunoglobulin, and chromosomes of the two lines indicated that one of them originated from leukemic cells while the other arose from residual normal blood cells. The morphology and capacity for immunoglobulin secretion in the line that arose from leukemic cells were similar to those found in EVB-carrying lymphoblastoid cell lines grown from patients without neoplasia and differed from those seen in fresh chronic lymphocytic leukemia cells. These observations suggest that the introduction of EBV into the leukemic cells may have caused them to differentiate in a fashion similar to that noted in normal B cells after exposure to EBV.     

CD39 expression on T lymphocytes correlates with severity of disease in patients with chronic lymphocytic leukemia

IntroductionChronic lymphocytic leukemia (CLL) is a B-cell disorder, but it is also associated with abnormalities in T-lymphocyte function. In this study we examine changes in T-lymphocyte CD39 and CD73 expression in patients with CLL.MethodsBlood samples were drawn from 34 patients with CLL and 31 controls. The cells were stained for CD3, CD4, CD8, CD19, CD39, and CD73 and analyzed by flow cytometry.ResultsOverall, patients with CLL had a higher percentage of CD39⁺ T lymphocytes than did controls. The percentage of cells expressing CD39 was higher in both CD4⁺ cells and CD8⁺ cells. Higher CD3/CD39 expression was associated with a later disease stage. No correlations between T-lymphocyte CD39 levels and CD38 or Zap-70 expression were observed. In contrast, the percentage of T lymphocytes and B lymphocytes that expressed CD73 was decreased in patients with CLL. Average B-lymphocyte CD73 expression was decreased in CLL because the majority of CLL clones were CD73. However a minority of CLL clones were CD73⁺, and patients with CD73⁺ clones tended to have earlier stage disease.ConclusionT-lymphocyte CD39 and CD73 expression may be useful prognostic markers in patients with CLL. Expression of CD73 on the malignant cell population in CLL may be a marker of better prognosis.     

Prodigiosin induces apoptosis of B and T cells from B-cell chronic lymphocytic leukemia.

We have previously reported that prodigiosin (2-methyl-3-pentyl-6-methoxyprodigiosene) induces apoptosis in human hematopoietic cancer cell lines with no marked toxicity in nonmalignant cell lines. In this study, we demonstrate that prodigiosin induces apoptosis of B-cell chronic lymphocytic leukemia (B-CLL) cells (n=32 patients). The dose-response for the cytotoxic effect of prodigiosin was analyzed in cells from 12 patients showing an IC(50) of 116+/-25 nM. Prodigiosin induced apoptosis of B-CLL cells through caspase activation. We also analyzed the cytotoxic effect of prodigiosin in T cells from B-CLL samples and no differences were observed with respect to leukemia cells. This is the first report showing that prodigiosin induces apoptosis in human primary cancer cells.