Biomedical subjects
Publications and source records attributed to M Massaia.
Preparations enriched in human blood B lymphocytes are able to take up polyamines efficiently. Uptake by T cells is barely detectable. Human non-circulating B cells (from tonsils) have a much lower ability to take up polyamines, as do mixed populations of bovine lymph nodes. B cells contain a higher amount of endogenous polyamines and show higher ornithine decarboxylase activity than T cells.
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Interaction of the glycosyl phosphatidylinositol-linked differentiation Ag CD73 (ecto-5'-nucleotidase) with the CD73-specific mAb 1E9 generates agonistic signals that strongly synergize with T cell activation induced by CD3 and CD2 mAb. This synergy is observed only when 1E9 is immobilized on plastic and occurs in the absence of accessory cells or exogenous lymphokines. 1E9 induces a rapid (though transient) increase in [Ca2+]i in a minor proportion (20 to 30%) of unfractionated T lymphocytes (presumably CD73+ cells). However, this [Ca2+]i mobilization is not sufficient to fully activate CD73+ T cells, as shown by the requirement of additional signals such as CD3 or CD2 stimulation to initiate T cell proliferation. These signals cannot be substituted by the exogenous lymphokines, rIL-1, rIL-2, or rIL-4, or PMA (when T cells are rigorously depleted of monocytes). These data indicate that CD73 may behave as an accessory molecule regulating interactions between T cells and antigens or APC. A comparison was carried out with mAb 9.3 to the differentiation Ag CD28, another agonistic molecule with activating properties similar to CD73. Despite their lower percentage, the ability of CD73+ T cells to amplify the proliferation induced by CD3 or CD2 mAb was equivalent or even greater than that of CD28+ T cells. Once activated, CD73+ cells may recruit the remaining (CD73-) cells primed by CD3 or CD2 stimulation. Based on these data, we suggest that CD73+ T lymphocytes may be a specialized subset to amplify immune responses originated by the CD3 and CD2 activation pathways. Finally, the functional association between CD73 and integral membrane molecules like CD3 and CD2 suggests that GPI-anchored molecules may play a role in transmembrane signaling mediated by conventional second messenger systems.
Tumor necrosis factor-alpha (TNF) is a cytokine that displays a pleomorphic array of effects on different cell populations. Evidence is presented that TNF may be constitutively produced by B-cell chronic lymphocytic leukemia (B-CLL) and hairy cell leukemia (HCL) cells and that it may play a relevant role in these diseases. These conclusions are based on the presence of circulating levels of TNF in the serum of 20 of the 24 patients tested (83.3%), while undetectable values were found in normal sera. The suggestion that the increased serum levels were due to the leukemic cell population is strengthened by the evidence that purified B-CLL and HCL cells may constitutively release variable degrees of TNF. These levels markedly increase after incubation with interferon gamma or phytohemagglutinin (PHA) plus phorbol myristate acetate (PMA). The cellular release of TNF by primary B-CLL cells was significantly (P less than .001) higher in B-CLL stage O-I patients compared with stage II-III patients. The demonstration that, in B-cell chronic lymphoproliferative disorders, the pathologic cells may release TNF was further confirmed by the presence of the mRNA for this cytokine in primary and/or in pre-activated cells. Recombinant TNF was capable of inducing a proliferative signal only in a minority of cases (4/24); in most cases it was ineffective, and, in a few, it reduced the degree of proliferation. Furthermore, in costimulatory experiments with interleukin-2 and PHA plus PMA, TNF was ineffective. On the other hand, when primary B-CLL cells were incubated in the presence of an anti-TNF antibody, in 8 of 12 independent experiments a 2- to 15-fold increase in thymidine uptake was documented. Taken together, these results suggest that TNF may play a regulatory role in the progression of the neoplastic clone in B-cell chronic lymphoproliferative disorders and may be implicated in some of the side effects associated with these diseases.
The recombinant interleukin-2 (rIL-2) generation of lymphokine-activated killer (LAK) cells was investigated in peripheral blood T lymphocytes (PBT) of 16 patients with monoclonal gammopathy of undetermined significance (MGUS) and 32 patients with multiple myeloma (MM). LAK activity was significantly decreased in MM, but not in MGUS patients, and was partially recovered in MM in the remission phase. This finding was unexpected, because CD8+ CD11b+ cells, which contain LAK precursors, are significantly increased in MM. LAK activity was investigated in purified CD8+ CD11b+ lymphocytes to discriminate between an intrinsic defect or a defective regulation by other T cell subsets. These cells were intrinsically unable to generate LAK activity fully following rIL-2 stimulation. MM showed the more pronounced LAK deficiency, while MGUS patients showed intermediate values. Phenotyping revealed significantly increased proportions of Leu7+ and HLA-DR+ cells in MM patients. These data reveal another dysregulation of T cell effector functions in patients with monoclonal gammopathies and offer further evidence of the impairment of their cell-mediated immunity.
A panel of monoclonal antibodies to the 69 kDa glycosyl phosphatidylinositol anchored lymphocyte differentiation antigen ecto-5'-nucleotidase (ecto-5'-NT, CD73) was produced using highly purified human placental 5'-NT as immunogen. Antibodies 1E9.28.1 and 7G2.2.11 inhibit soluble placental 5'-NT activity and recognize lymphocyte CD73 in indirect immunofluorescence and immunoprecipitation assays. In addition, 1E9.28.1 induces vigorous T cell proliferation in the presence of submitogenic doses of phorbol myristate and F(ab')2 goat anti-mouse Ig. Both antibodies can be used to purify the three major forms of placental 5'-NT by affinity chromatography. By two-color immunofluorescence, CD73 was found to be expressed on 19 +/- 5% of CD3+, 11 +/- 4% of CD4+, 51 +/- 14% of CD8+, 25 +/- 8% of CD28+, 15 +/- 5% of CD29+, 27 +/- 7% of CD45RA+, and 70 +/- 6% of CD19+ lymphocytes. Within T cells, CD73 expression is restricted to the CD28+ subset. Thus, CD73 is found on subsets of both T and B lymphocytes, with the highest expression on B cells and CD8+ T cells. In sections of hyperplastic tonsil, CD73 expression is restricted to the small lymphocytes of the follicular mantle zone, a small subset of extrafollicular lymphocytes situated within the epithelium of the tonsillar crypt, and to follicular dendritic cells within the lower part of the "light-zone." CD73 is also detected on subsets of endothelial cells of capillaries and venules and the basal layer of non-keratinizing squamous epithelium and transitional cell type mucosa of many tissues. Given the tissue distribution of CD73, along with its glycosyl phosphatidylinositol membrane anchoring and the observation that some CD73 antibodies are mitogenic, we propose that this interesting antigen may play a role in cell activation, lymphocyte homing, and/or cell adhesion.
The expression of three growth-regulated protooncogenes, c-myc, c-myb, and p53, and the S-phase-specific histone H3 gene, was compared in bone marrow cells from multiple myeloma patients and normal controls by measuring the amount of specific RNA by Northern blot analysis. Four samples contained at least 72% of myeloma cells, one sample 43%, and one 11%. Expression of the protooncogenes was similar in normal and myeloma bone marrow cells, whereas that of histone H3 gene was significantly reduced (between 10 and 15 times) in samples containing at least 43% of malignant plasma cells and not detectable in those containing more than 72% of neoplastic cells. Protooncogene levels of expression were compared to those of the H3 gene to distinguish the increased expression of a growth-regulated gene due to a true deregulation from overexpression reflecting solely an increase in the fraction of cycling cells. The ratios of expression of protooncogenes to histone H3 were markedly increased in multiple myeloma cells; the highest ratios were found in the patients with the highest number of malignant plasma cells. These results suggest that the expression of three growth-regulated oncogenes (c-myc, c-myb, p53) is altered in myelomatous plasma cells.
We analyzed the immunoglobulin (Ig) heavy chain gene rearrangement in the peripheral blood lymphocytes of a patient with multiple myeloma (MM). Although the morphological and immunological examination did not reveal the presence of circulating plasma cells, a monoclonal Ig gene rearrangement was detected. This observation indicates that a monoclonal expansion of circulating B cells was present in the peripheral lymphocytes of this patient.
CD3+CD8+ cells, purified from peripheral blood T cells by depletion of CD4+ lymphocytes, were tested for their cytotoxic activity against K-562 or HL-60 cells. Freshly prepared cells had no cytotoxic function, but upon exposure to recombinant interleukin 2 (rIL2) in vitro they acquired a lymphokine-activated killer (LAK) activity. Fractionation of CD3+CD8+ cells into CD11b+ and CD11b- cells demonstrated that all the cells with the potential of developing LAK cell functions were within the CD8+CD11b+ subset. These cells lacked natural killer cell markers such as CD16 or NKH1, but a proportion of them stained for Leu-7. Furthermore, they expressed the alpha/beta chains, but not the gamma/delta chains, of the T cell receptor, as could be determined by staining with the appropriate monoclonal antibodies. CD8+CD11b+ cells had a large granular lymphocyte morphology, as shown by both cytochemical and electron microscopy analyses. They proliferated in response to IL2 in vitro and developed cytotoxic functions against a number of natural killer resistant targets. Their response to phytohemagglutinin or pokeweed mitogen was very weak or absent. By contrast, CD8+CD11b- cells failed to generate LAK cells in response to rIL2, did not show a large granular lymphocyte morphology, but responded vigorously to phytohemagglutinin or pokeweed mitogen. Purified CD8+CD11b+ cells were cloned by limiting dilution in the presence of rIL2. The observed cloning efficiency of 19 +/- 0.3% indicates that a fraction of the cells only could respond to IL2. Furthermore, only 50% of the clones obtained had a LAK cell function. These data show that CD8+CD11b+ cells represent a heterogeneous cell population. Nevertheless this cell subset probably represents the major source of LAK cell progenitors within the circulating T cells.
We studied the ability of chronic lymphocytic leukemia (CLL) B cells to stimulate proliferation and major histocompatibility complex (MHC)-unrestricted [natural killer (NK)-like] cytotoxicity in a mixed lymphocyte tumor-cell culture (MLTC). CLL-derived B cells induced a significantly higher proliferative response than did B cells from healthy normal donors. Comparable levels of interferon and interleukin-2 production were detected in the mixed lymphocyte cultures (MLC) set up with normal B cells from healthy donors and in MLTC. Higher levels of NK-like cytotoxic activity were induced after stimulation with CLL than with normal B cells in nonlytic precursors. Inhibition experiments with specific monoclonal antibodies indicate that the NK-like response in MLTC is attributable to HLA class II antigens, which are expressed at comparable levels on CLL and normal B cells. Percoll gradient centrifugation of the MLC and MLTC recovered cells demonstrated that the NK-like effectors in both types of cultures were blast-transformed, highly mitotic cells.
Bone marrow plasma cell proliferative activity has been evaluated in a large series of multiple myeloma (MM) patients. This kinetic parameter has been shown to be a useful tool for patient management, and contributes to a correct diagnosis and a selection of high-risk patients who can be offered high-dose chemotherapy. The role of ras oncogenes has been evaluated in the pathogenesis of MM. A point-mutated and activated H-ras oncogene, introduced in a human lymphoblastoid cell line, was able to induce neoplastic transformation and differentiation to plasma cell. Indeed, mutated alleles of ras genes have been detected in a high percentage of myeloma patients in relapse phase. Phenotypical and functional studies have been carried out in T-lymphocyte subsets and an impaired cellular immunity has been detected. Such an impairment was related to the disease status: marked alterations were detected in relapse phase, whereas a partial recovery was observed during remission phase.
Serum beta-2-microglobulin (beta 2M) has been suggested as the most powerful prognostic factor in multiple myeloma (MM). This paper investigates its ability to detect remission and relapse in individual patients. A correlation analysis was carried out between beta 2M and M component determinations, at diagnosis and monthly during follow-up in 21 consecutive MM patients with normal renal function. A statistically significant correlation was observed in 52.4% only. The lack of correlation in the remaining cases was due to low beta 2M production at diagnosis, or independent fluctuation of these 2 parameters. Serum beta 2M proved a much less reliable parameter for the detection of tumour variations than simpler M-component determination.
In order to assess the prognostic value of rapid tumor mass reduction in responding multiple myeloma (MM) patients, 100 consecutive patients were analyzed, and bone marrow plasma cell kinetic characteristics were evaluated at diagnosis. Forty-two patients obtained a tumor mass reduction greater than or equal to 50% with three cycles of chemotherapy and within 3 months (early responder myeloma [ERM]), and 23 in greater than 3 months (slow responder myeloma [SRM]). Survival rates in these two groups were not statistically different (P = .07). The labeling index (LI) of bone marrow plasma cells was significantly higher in ERM patients than in SRM patients (1.8 +/- 2.0 v 0.8 +/- 0.7, P = .006). The LI was used to separate the ERM patients into two well-defined subgroups. ERM patients with a LI greater than or equal to 2% showed a median survival of 16.4 months, whereas ERM patients with a LI less than 2% did not reach the median survival at 46.9 months (P less than .0044). Remission duration was also significantly different: 12.2 months in the high LI subgroup and 26.3 months in the low LI subgroup (P less than .0025). Early response itself does not correspond to shorter remission duration and shorter survival, but it is a poor prognostic factor if associated with a high plasma cell proliferative activity.
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The characteristics of a human cell line (LP-1) derived from the peripheral blood of a patient with IgG-lambda myeloma in leukemic transformation are described. The cells resemble immature plasma cells in that they exhibit a membrane phenotype that is intermediate between late B lymphocytes and plasma cells, even though they secrete IgG-lambda chains. Treatment of LP-1 cells with 12-0 tetradecanoylphorbol-13-acetate (TPA) or pokeweek mitogen (PWM) induces the appearance of surface markers and ultrastructural features typical of mature plasma cells but does not affect their proliferative activity. Molecular analysis of the cell line showed an increased expression of the c-myc protooncogene and the presence of abnormally sized transcripts. Conventional cytogenetics and pulsed-field gel electrophoresis showed no structural rearrangements of the c-myc gene, suggesting that the abnormal c-myc expression may be due to point mutations or small deletions within the gene. The LP-1 cell line is a useful model in which to study the process of B-cell maturation; such study may lead to the uncovering of unusual mechanisms of c-myc activation. Furthermore, the LP-1 cell is a potential partner in the generation of human hybridomas.
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Ecto-5'-nucleotidase (ecto-5'NT) activity is highly expressed by CTL precursor cells. The aim of this work was to investigate whether ecto-5'NT is involved in their functional activation. The inhibition of ecto-5'NT activity by biochemical (alpha,beta-methyleneadenosine 5-diphosphate; AOPCP) or immunologic inhibitors (polyclonal anti-ecto-5'NT serum) suppressed the proliferative and cytotoxic activation of CTL against a lymphoblastic B cell line in primary one-way MLC. The kinetic analysis of suppression showed that AOPCP and anti-5'NT serum had different effects: AOPCP suppressed only the activation of the cytotoxic program whereas anti-5'NT serum suppressed also recognition and binding to the stimulatory/target cell. Inhibition of CTL generation was not a metabolic consequence of the purine salvage blockade. Incubation of AOPCP-treated MLC with hypoxanthine restored proliferation but not cytotoxicity. The ecto-5'NT inhibitors used had no general toxic effect on cell numbers or viability. AOPCP selectively affected CTL generation and displayed no activity against mitogen-induced proliferation, activation of Ts cells, and generation of IL-2-activated killer cells. These data indicate that ecto-5'NT has a critical role in the functional activation of alloreactive CTL.
Peripheral blood lymphocytes from 9 monoclonal gammopathies of undetermined significance (MGUS) and 27 multiple myelomas (MM) were studied with a panel of monoclonal antibodies (MoAb) that recognize B and T lymphocytes and plasma cells. No difference in the percentage of B lymphocytes, identified by B1 and B4 MoAb, was observed in MGUS and MM patients versus normal controls. However, high percentages of circulating lymphocytes expressing plasma cell-associated antigens were detected in MM (HAN-PC1+ = 29.4 +/- 20.4%; TEC-T10+ = 27.8 +/- 19.2%) whereas they were in the normal range in MGUS (HAN-PC1+ = 8.8 +/- 5.8% p = 0.006; TEC-T10+ = 5.7 +/- 4.7% p less than 0.001). Almost identical results were obtained using PCA-1 MoAb in 17 of these patients. TEC-T10+ and PCA-1+ lymphocytes were sorted and re-analyzed with phycoerythrin conjugated MoAb in 3 healthy subjects, 2 MGUS, and 4 MM patients. In normal subjects and in MGUS the majority of PCA-1+ cells belonged to the B lineage (Leu 2-, Leu3-, Leu 15-, HLA-Dr+), whereas the majority of TEC-T10+ cells are represented by activated T cells and NK cells (Leu 15+). In MM an abnormal expansion of T lymphocytes was chiefly responsible for the high values of lymphocytes expressing plasma cell-associated antigens. Moreover, in MM a clinical evaluation showed a correlation between the presence of these lymphocytes and an aggressive disease. Indeed, they can be considered a useful prognostic marker.