CD1d is expressed on B-chronic lymphocytic leukemia cells and mediates alpha-galactosylceramide presentation to natural killer T lymphocytes

Generation of immune responses against B cell chronic lymphocytic leukemia (B-CLL) has been the aim of several studies that have demonstrated a poor antigen presenting ability of B-CLL cells and an inconsistent emergence of T cells capable of killing efficiently the leukemic cells. CD1d is a restriction element structurally related to the major histocompatibility complex (MHC) and capable of presenting lipid antigens to CD1d-restricted T cells (also defined as natural killer-T [NKT] cells). The synthetic lipid alpha-galactosylceramide (alpha-GalCer) has been characterized as a potent stimulator of CD1d-restricted T cells. We have investigated the expression of CD1d on B-CLL cells. CD1d was detected by flow cytometric analyses on leukemic cells of all B-CLL cases studied (n = 38) and was expressed at higher density on cells carrying unmutated immunoglobulin variable region (IgV) genes. In addition, CD1d on B-CLL cells mediated the presentation of alpha-GalCer to CD1d-restricted T cells, which in turn induced B-CLL cell death. At variance with another study (Metelitsa et al., Leukemia 2003;17:1068-77), no correlation between expression levels of CD1d and susceptibility to NKT cell lysis was observed. Proliferation and production of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) by CD1d-restricted T cells, in the presence of B-CLL cells loaded with alpha-GalCer, were also observed. Our study demonstrates that B-CLL cells express a monomorphic restriction element that is functionally capable of antigen presentation and can be useful to design novel B-CLL immunotherapies.     

Colony-forming assay for circulating chronic lymphocytic leukemia cells

In these studies, we report adaptation of a colony-forming assay to chronic lymphocytic leukemia (CLL) peripheral blood cells. T-lymphocyte-depleted CLL peripheral blood cells were cultured with irradiated, normal T cells and media conditioned by normal, mitogen-stimulated T cells in methylcellulose. Colonies containing small and transformed lymphocytes appeared after 5–7 days incubation. The plating efficiency of CLL colonies was 0.15 ± 0.08% ($\text{x}\text{±}\text{S.D.}$), similar to that of other colony-forming systems. The majority of CLL colony-forming cells were in S phase (50 ± 4%, $\text{x}\text{±}\text{S.E.}$) as determined by thymidine suicide and the fraction of colony-forming cells in S phase was inversely related to the WBC. Cells harvested from CLL colonies lacked surface markers for T lymphocytes and stained positively for monoclonal surface and/or cytoplasmic immunoglobulin light chains. A 1-h incubation was used to study the in vitro response of CLL colony-forming cells to adriamycin and melphalan. Preliminary studies suggest differences in patterns of in vitro sensitivity to melphalan between patients previously treated with alkylating agents and those who had not received treatment. This system can be used to study regulation of CLL cell proliferation, and may have utility in predicting response to chemotherapeutic agents.     

Acute myeloid leukemia and lung cancer occurring in a chronic lymphocytic leukemia patient treated with fludarabine and autologous peripheral blood stem-cell transplantation

An increased incidence of different malignancies associated to chroniclymphocytic leukemia (CLL) has been reported. The association of CLL and acuteleukemia is a rare event described in <1% of CLL, the type of acuteleukemia being either from the lymphoid or more often from the myeloidlineage. The coexistence of acute myeloid leukemia (AML) and CLL in the samepatient has been occasionally reported. Most of these cases have beenassociated with the administration of chemotherapy or radioterapy for CLL,suggesting that the former may be a secondary leukemia. On the other hand, CLLcould precede, but could also be diagnosed at the same, or delayed time asAML, suggesting the presence of other leukemogenic factors. We describe theexceptional development of AML and lung cancer in a patient with previouslydiagnosed CLL in minimal residual disease status after fludarabine treatmentfollowed by autologous peripheral blood stem-cell transplantation.     

Monoclonal origin of B cells producing k, λ and kλ immunoglobulin light chains in a patient with chronic lymphocytic leukemia

Immunoglobulin gene rearrangements can be used as genetic markers of clonality in the study of B-cell populations [4]. We have therefore analysed the structure and expression of heavy and light chain immunoglobulin genes in lymphocytes of a patient with chronic lymphocytic leukemia, where we found both k and λ producing B cells, but in most of the cells both k and λ chains were co-expressed on the same surface membrane. Single rearrangements were observed in μ, JH, k and λ DNA sequences, thus providing strong evidence for the monoclonal origin of the cells bearing different light chains. Moreover, the analysis of Ig sequence RNA showed, in addition to normal μ, k and λ mRNA molecules, high levels of a small λ related RNA sequence. These findings are discussed in relation to a model of B-lymphocyte differentiation which could be either an additional or an alternative hypothesis to the current one of isotypic exclusion.     

Enzymological studies in chronic lymphocytic leukemia

Adenosine deaminase (ADA), 5'nucleotidase (5'NT), ecto-5'NT, purine nucleoside phosphorylase (PNP), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenine phosphoribosyltransferase (APRT), adenosine kinase (AK), AMP-deaminase (AMPD) and adenylate kinase (AdKin) activities were assayed in peripheral blood lymphoid cells from 20 patients with B-cell type chronic lymphocytic leukemia (CLL). Significantly decreased mean activities of ADA, 5'NT, ecto-5'NT, PNP and AMPD were observed when comparing B-CLL lymphoid cells with control peripheral blood lymphocytes (PBL). AK and AdKin activities however, were found to be higher in B-CLL. Relatively wide ranges of ADA and 5'NT activity were observed. In patients with paraproteinaemia, 5'NT activity was found to be relatively high and in the range of the activities in normal PBL. ADA activity seemed to be slightly higher in patients without paraproteinaemia. No correlation could be found between the enzyme activities and the number of cells rosetting with sheep erythrocytes or bearing surface immunoglobulin (sIg). A relationship was suggested between 5'NT activity and Ig production.     

PHA-ICC, ADCC and NK in patients with ANLL in CR: human fibroblastic interferon fails to increase NK-active cell frequency

PHA-ICC, ADCC and NK activity of PBL were studied in ten patients with ANLL in CR and in eighteen normal controls in the presence and absence of HFIF. No statistically significant differences were recorded among the two groups with regard to basic lymphocyte functions. Although the parameters of lymphocyte function remained analogous for those tested, the analysis at the single cell level revealed that HFIF stimulation increases the number of NK active cells and target binding cells among normals, but not in leukemic patients.     

Membrane markers,karyotypic abnormalities,ultrastructure and functional properties of lymphocytes in a case of ‘D-cell’ chronic lymphatic leukemia

D cells are lymphocytes bearing both receptors for the third complement component and the ability to form spontaneous rosettes with SRBC. We report the case of a patient with a D-cell chronic lymphatic leukemia who presented a long evolution without treatment and whose leukemic cell characteristics have been extensively studied. Cytogenetic analysis showed numerous karyotypic abnormalities among leukemic cells; all metaphases were hypodiploid and arranged in four different clones; seven marker chromosomes were present. The cells were found to bear human T-cell specific antigen, the T helper/inducer phenotype, HLA-A and HLA-B determinants, but no HLA-DR antigens. They displayed a high proliferative response to PHA and Con A, no response to PWM stimulation, and possibly the capacity of allogeneic stimulation in the mixed lymphocyte culture system. Assays for cell-mediated cytotoxicity in the CML system, and for K and NK activities were negative.     

Phorbol ester-induced loss of colchicine ultrasensitivity in chronic lymphocytic leukaemia lymphocytes

On exposure to the phorbol ester 12-O-tetradecanoyl-13-acetate (TPA) the pathological (non-dividing) lymphocytes of B-cell chronic lymphocytic leukaemia (CLL) lose their characteristic ultrasensitivity to the cytocidal action of colchicine in vitro. They are no longer killed in 1 day by the drug at 10(-6)M-concentration. The effect was the same whether the cells were incubated in the continuous presence of TPA, or subjected instead to pulse-treatment with it (for as little as 5 min.). Colchicine at one thousand times greater concentration was now needed to kill the cells. CLL lymphocytes already primed to undergo interphase death by pretreatment with colchicine could be prevented from doing so by early addition of TPA. A marked proportion of those CLL lymphocytes destined to undergo early spontaneous death in vitro in the absence of colchicine could be prevented from doing so by TPA. The loss of colchicine ultrasensitivity applied to cells which had not yet undergone TPA-induced morphological transformation to blast-like cells or differentiation to cells containing abundant cytoplasmic immunoglobulins (CIg). These transformed cells materialised in greatest incidence (70-80%) after 3 days of culture, an observation in agreement with others workers.     

Leukemia of T-helper lymphocytes: Clinical and functional features

Leukemia of T-lymphocytes is rare. Chronic and subacute forms of T-cell leukemia have been described including Sézary cell leukemia, T-cell leukemia of Japan, and rare cases of chronic lymphocytic leukemia, prolymphocytic leukemia and hairy cell leukemia. Several studies have analyzed the functional capacity of lymphocytes from patients with T-cell leukemias. In many cases Sézary cells appear to be “helper” T-cells which cooperate with B-lymphocytes in immunoglobulin synthesis. In contrast, lymphocytes from the Japanese form of T-cell leukemia have been reported to function as suppressor cells. We describe an unusual leukemia in a 40-year old man, whose clinical course was characterized by fever, jaundice, hepatosplenomegaly, anemia and neutropenia. Lymph node enlargement and skin infiltration were notably absent. The leukemic cells were atypical mononuclear cells with a high nuclear-cytoplasmic ratio and convoluted nuclei. Immunologic studies of these cells demonstrated them to be T-lymphocytes with receptors for sheep erythrocytes. They reacted with an anti-thymocyte serum and responded to T-cell mitogens and to alloantigens. The leukemic T-cells had helper activity and could cooperate with B-lymphocytes in pokeweed mitogen (PWM) stimulated immunoglobulin synthesis. Concanavalin-A (Con-A) inducible suppressor cell activity was absent. The clinical and immune studies suggest that this case represents a previously undescribed form of T-cell leukemia.     

Accessory role of autologous T lymphocytes and adherent cells for the in vitro proliferation of T-cell colony-forming cells from patients with T-cell acute lymphoblastic leukemia

Peripheral blood T colony-forming cells (T-CFC) from patients with T-cell malignancies can proliferate in methylcellulose in the absence of added growth factors or mitogenic stimulation. Mononuclear cells (MNC) from 7 patients with T-cell acute lymphoblastic leukemia were separated into cells forming rosettes with sheep erythrocytes (E+) or not (E-). E- cells were further depleted by complement-mediated cytotoxicity with OKT3 monoclonal antibody (E-OKT3- cells). The study of their spontaneous T-cell colony-forming ability suggested that proliferation of T-CFC in the absence of added growth factors requires cellular cooperation because: (1) No colony growth was observed at low cell concentrations (up to 2 X 10(4) cells/ml) whereas at higher cell densities the number of colonies increased exponentially; (2) The plating efficiency from unfractionated MNC was higher than that from E-OKT3- or E+ cells. Irradiated autologous E+ cells enhanced the plating efficiency from blast-enriched cell fractions (E-OKT3-) when co-cultured either directly in methylcellulose or separately in a two layer assay (agar-methylcellulose), suggesting that their activity could be due to diffusible factors; (3) Adherent-cell depletion of MNC decreased colony formation. Autologous irradiated adherent cells were able to restore the plating efficiency from MNCA- cells when co-cultured directly in methylcellulose but not in separate layers; however, media conditioned by patients' A+ cells could enhance the colony growth from patients' MNCA- cells, indicating that their activity could also be mediated by constitutively released soluble factors.     

The correlation of adenosine deaminase and purine nucleoside phosphorylase activities in human lymphocytes subpopulations and in various lymphoid malignancies

Adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) were measured in normal human and in malignant lymphoid cells. Thymocytes had high ADA activity (21.2 +/- 6.8 10(3) nM/h/mg) and low PNP activity (1.2 +/- 0.6), whereas T peripheral blood lymphocytes (PBL) had low ADA activity (1.20 +/- 0.22) and high PNP activity (2.8 +/- 1.3). Moreover cortico-thymocytes had higher ADA and lower PNP levels than medullary thymocytes. A linear correlation was observed between ADA and PNP activities in both thymocytes and T-PBL. Cells from 13 patients with T acute lymphoblastic leukemia (ALL) and 10 patients with T lymphoblastic lymphoma (LL) had very high levels of ADA (respectively 13.0 +/- 5.4 and 22.8 +/- 14) and low levels of PNP (respectively 1.9 +/- 0.8 and 2.5 +/- 1.4). However no clear relationship appeared between subgroups of these T-cell malignancies defined by their patterns of surface antigens, revealed by reactivity with monoclonal antibodies, and ADA and PNP levels, and there was no correlation between the two enzymes. In contrast, cells from 31 patients with HLA-DR+ common ALL had significantly low values of ADA as compared to cells from six patients with HLA-DR- common ALL and a linear correlation was observed between ADA and PNP in cells from children with non-T, non-B ALL. These results show that specific stages of T-cell development may be characterized by the relationships and the correlation between the two enzymes and suggest that T-ALL and T-LL appear to be the group of lymphoid malignancies with a high degree of incoordination between ADA and PNP activities.     

Cyclophosphamide,alvocidib (flavopiridol), and rituximab,a novel feasible chemoimmunotherapy regimen for patients with high-risk chronic lymphocytic leukemia

Alvocidib has demonstrated efficacy in high-risk chronic lymphocytic leukemia (CLL) patients. In this phase I study, we combined cyclophosphamide, alvocidib and rituximab (CAR) in a schema designed to mitigate tumor lysis syndrome (TLS) seen previously with alvocidib. Nine nucleoside analog-naïve, high-risk patients received escalating doses of CAR therapy. Dose limiting toxicity was not experienced. No instances of TLS were observed. Patient responses included three complete remissions and four partial remissions. CAR was tolerable and active in high-risk CLL patients without TLS toxicity. With continued monitoring of toxicities, a phase Ib/II study of this combination as frontline therapy is warranted.