M Ratta

University of Bologna, Bologna, Emilia-Romagna, Italy

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Publications (25)123.57 Total impact

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    ABSTRACT: An anti-CD38 mAb (IB4) coupled to saporin-S6, a type 1 ribosome-inactivating protein (RIP), was designed for ex vivo or loco-regional therapeutical applications in myeloma and lymphoma. The ability of this immunotoxin to eliminate CD38+ cells was studied in vitro on selected CD38+ human cell lines (Raji, HBL6, L540 and CEM) and on CD38+ neoplastic cells from a Non Hodgkin Lymphoma (NHL) patient. HBL6, Raji and L540 cells resulted very sensitive to the IB4/saporin-S6 conjugate, concentrations as low as 100 pM of the immunotoxin completely inhibited protein synthesis. CD38+ neoplastic cells from the NHL patient were completely eliminated after treatment with immunotoxin at 10 nM concentration. CFU-c rescue by bone marrow precursors was maintained after exposure to the immunotoxin. These results indicate that IB4/saporin-S6 is endowed with strong and specific cytotoxic effects on selected CD38+ tumor cells lineages. Consequently, it is reasonable to propose a clinical use of the IB4/saporin-S6 for ex vivo purging of unwanted cells (e.g. depletion of contaminating neoplastic cells in aphereses obtained from G-CSF-treated patients) or for loco-regional therapies of CD38+ tumors.
    Journal of biological regulators and homeostatic agents 01/2005; 19(3-4):145-52. · 5.18 Impact Factor
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    ABSTRACT: Peripheral blood CD14+ monocytes from multiple myeloma (MM) patients can be induced to differentiate into fully functional, mature, CD83+ dendritic cells (DCs) which are highly efficient in priming autologous T lymphocytes in response to the patient-specific tumor idiotype (Id). We have recently scaled up our manufacturing protocol for application in a phase I-II clinical trial of anti-Id vaccination with DCs in MM patients. Elegible patients received a series of by-monthly immunizations consisting of three subcutaneous and two intravenous injections of Id-keyhole limpet hemocyanin (KLH)-pulsed DCs (5 x -, 10 x -, 50 x 10(6) cells and 10 x -, 50 x 10(6) cells, respectively). To generate DCs, monocytes were labeled with clinical grade anti-CD14 conjugates and positively selected by immunomagnetic separation. Cells were then cultured, according to Good Manufacturing Practice guidelines, in FCS-free medium in cell culture bags, and differentiated to DCs with GM-CSF plus IL-4 followed by TNF-alpha or, more recently, by a cocktail of IL-1beta, IL-6, TNF-alpha and prostaglandin-E2. Before maturation, Mo-DCs were pulsed with the autologous Id as whole protein or Id (VDJ)-derived HLA class I restricted peptides. Ten MM patients, who had been treated with two courses of high-dose chemotherapy with peripheral blood stem cell support, entered into the clinical study. CD14+ monocytes were enriched from 16.1+/-5.7% to 95.5+/-3.2% (recovery 67.9+/-15%, viability > 97%). After cell culture, phenotypic analysis showed that 89.6+/-6.6% of the cells were mature DCs. We obtained 2.89+/-1 x 10(8) DCs/leukapheresis which represented 24.5+/-9% of the initial number of CD14+ cells. Notably, the cytokine cocktail induced a significantly higher percentage and yield (31+/-10.9 of initial CD14+ cells) of DCs than TNF-alpha alone, secretion of larger amounts of IL-12, potent stimulatory activity on allogeneic and autologous T cells. Storage in liquid nitrogen did not modify the phenotype or functional characteristics of pre-loaded DCs. The recovery of thawed, viable DCs, was 78+/-10%. Thus, positive selection of CD14+ monocytes allows the generation of a uniform population of mature pre-loaded DCs which can be cryopreserved with no effects on phenotype and function and are suitable for clinical trials. Based on these results, a DCs-based phase II trial of anti-Id vaccination with VDJ-derived HLA class I-restricted peptides and KLH is underway for lymphoma patients.
    Leukemia and Lymphoma 08/2004; 45(7):1419-28. · 2.61 Impact Factor
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    ABSTRACT: The expression of cytotoxic T-lymphocyte antigen-4 (CTLA-4) molecule in human normal and neoplastic hematopoietic cells, both on the cell membrane and in the intracellular compartment, was evaluated. Flow cytometric analysis carried out with a panel of anti-CTLA-4 human single-chain fragment of variable domain (scFv) antibodies revealed that CTLA-4 was not expressed on the surface, whereas it was highly expressed within the cytoplasm, in freshly isolated peripheral blood mononuclear cells (PBMCs), T cells, B cells, CD34(+) stem cells, and granulocytes. Various treatments with agents able to specifically activate each cell type induced CTLA-4 expression on the surface of these cells. Similarly, increased CTLA-4 expression was observed in different hematopoietic cell lines although they also expressed surface CTLA-4, at different degrees of intensity, before activation. Surprisingly, CTLA-4 RNA transcripts were detectable in such cell lines only after nested polymerase chain reaction (PCR) specific for CTLA-4 extracellular domain, suggesting a very fast CTLA-4 RNA processing accompanied by prolonged CTLA-4 protein accumulation. We further demonstrated surface expression of CTLA-4 in a variety of acute and chronic myeloid leukemias (AMLs and CMLs) and B- and T-lymphoid leukemias, either adult or pediatric. CTLA-4 was expressed in 25% to 85% of AMLs and CMLs depending on the leukemia subtype and the epitope analyzed, whereas in acute B- and T-leukemias CTLA-4 expression was mainly cytoplasmic. Chronic B leukemias appeared to express CTLA-4, both on the surface and in cytoplasm, whereas few cases tested of chronic T leukemias were negative. Two anti-CTLA-4 immunotoxins (scFvs-saporin) induced in vitro apoptosis of neoplastic cells from a representative AML, suggesting a novel immunotherapeutic approach to AML based on CTLA-4 targeting.
    Blood 02/2003; 101(1):202-9. · 9.78 Impact Factor
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    ABSTRACT: We studied concentration, phenotype, and function of peripheral blood (PB) dendritic cells (DCs) from patients with multiple myeloma (MM). The absolute number of circulating precursors of myeloid and plasmacytoid DCs was significantly lower in MM patients than in healthy subjects. After maturation, PBDCs from MM patients showed significantly lower expression of HLA-DR, CD40, and CD80 antigens and impaired induction of allogeneic T-cell proliferation compared with controls. Remarkably, they were not capable of presenting the patient-specific tumor idiotype to autologous T cells. Conversely, DCs generated in vitro from CD14(+) monocytes from the same patients, and PBDCs freshly isolated from healthy donors efficiently stimulated allogeneic and autologous T cells. To clarify the mechanism of PBDC deficiency in MM, we investigated the effects of the main plasma cell growth factor, interleukin-6 (IL-6), on the development of DCs from CD34(+) cells. IL-6 inhibited the colony growth of CD34(+) DC progenitors and switched the commitment of CD34(+) cells from DCs to CD14(+) CD1a(-) CD86(-)CD80(-) CD40(+/-)HLA-DR +/- monocytic cells exerting potent phagocytic activity but no antigen-presentation capacity. This effect was reversed by anti-IL-6 antibodies. Growing CD34(+) cells in the presence of autologous serum (without IL-6) also suppressed the development of functional DCs. This study demonstrates that PBDCs from MM patients are functionally defective, partially because of IL-6-mediated inhibition of development. This brings into question the advisability of using PBDCs as antigen carriers for immunotherapy trials in MM. The results also suggest a novel mechanism whereby myeloma cells escape immune recognition.
    Blood 08/2002; 100(1):230-7. · 9.78 Impact Factor
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    ABSTRACT: We have recently shown that interleukin (IL-)11 induces polarization of human T-cells by inhibiting macrophage production of IL-12 and by exerting a direct effect on CD4+ T-cells. In this study, we investigated the effects of IL-11 on the kinetic activation and apoptosis of T-cell subsets stimulated with anti-CD3/CD28 antibodies, anti-CD3 and IL-2 or dendritic cells. Apoptosis and cell cycle analysis of T-cells were assessed by double staining with propidium iodide and intracellular Ki-67 and by acridine orange staining. The expression of the negative regulator of the cell cycle p27Kip1 (p27) was also determined by flow cytometry. Our results show that 18 hours of incubation with IL-11 resulted in a significantly higher number of cycling CD4+ cells, CD4+CD45RA+ naive T-cells and CD4+CD45RO+ memory T-cells, but not of CD8+ cells. The kinetic activity of IL-11 was observed up to 72 hours, when the peak value of S-phase cells occurred. IL-11 also significantly enhanced CD4+ and CD4+CD45RA+ cell proliferation when T-cells were co-incubated with allogeneic dendritic cells. Conversely, IL-11 did not protect any of the T-cell subsets from apoptosis. At the functional level, a type-2 cytokine pattern of cultured T-lymphocytes was observed after 5 days of incubation with IL-11. Proliferation and functional activation of T-cells were preceeded by downregulation of p27, which occurred as early as 12 hours after incubation with IL-11. IL-11 induces Th-2 polarization and cell-cycle entry of human CD4+, CD4+CD45RA+ and CD4+CD45RO+cells and their activation is associated with the downregulation of p27.
    Haematologica 05/2002; 87(4):373-80. · 5.94 Impact Factor
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    ABSTRACT: Exploration of the immunomodulatory activities of the multifunctional cytokine interleukin-11 (IL-11) has prompted several therapeutic applications. The immunomodulatory effects of IL-11 on human antigen-presenting cells and on T cells were investigated. IL-11 inhibited IL-12 production by activated CD14(+) monocytes, but not by mature dendritic cells (DCs) stimulated via CD40 ligation. Moreover, IL-11 did not affect either DC maturation, as demonstrated by phenotypic analysis and evaluation of cytokine production, or DC generation from progenitor cells in the presence of specific growth factors. Molecular analysis demonstrated the expression of IL-11 receptor messenger RNA in highly purified CD14(+) monocytes, CD19(+) B cells, CD8(+), and CD4(+) T cells, and CD4(+)CD45RA(+) naive T lymphocytes. In keeping with this finding, IL-11 directly prevented Th1 polarization of highly purified CD4(+)CD45RA(+) naive T cells stimulated with anti-CD3/CD28 antibodies, as demonstrated by significant increases of IL-4 and IL-5, by significantly decreased interferon-gamma production and by flow cytometry intracellular staining of cytokines. Coincubation of naive T cells with DCs, the most potent stimulators of Th1 differentiation, did not revert IL-11-mediated Th2 polarization. Furthermore, parallel experiments demonstrated that the activity of IL-11 was comparable with that induced by IL-4, the most effective Th2-polarizing cytokine. Taken together, these findings show that IL-11 inhibits Th1 polarization by exerting a direct effect on human T lymphocytes and by reducing IL-12 production by macrophages. Conversely, IL-11 does not exert any activity on DCs. This suggests that IL-11 could have therapeutic potential for diseases where Th1 responses play a dominant pathogenic role.
    Blood 06/2001; 97(9):2758-63. · 9.78 Impact Factor
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    ABSTRACT: We studied cytokine-driven differentiation of primitive human CD34(+)HLA-DR(-) cells to myeloid dendritic cells (DC). Hemopoietic cells were grown in long-term cultures in the presence of various combinations of early acting cytokines such as FLT3-ligand (FLT3-L) and stem cell factor (SCF) and the differentiating growth factors GM-CSF and TNF-alpha. Two weeks of incubation with GM-CSF and TNF-alpha generated fully functional DC. However, clonogenic assays demonstrated that CFU-DC did not survive beyond 1 wk in liquid culture regardless of whether FLT3-L and/or SCF were added. FLT3-L or SCF alone did not support DC maturation. However, the combination of the two early acting cytokines allowed a 100-fold expansion of CFU-DC for >1 month. Phenotypic analysis demonstrated the differentiation of CD34(+)DR(-) cells into CD34(-)CD33(+)DR(+)CD14(+) cells, which were intermediate progenitors capable of differentiating into functionally active DC upon further incubation with GM-CSF and TNF-alpha. As expected, GM-CSF and TNF-alpha generated DC from committed CD34(+)DR(+) cells. However, only SCF, with or without FLT3-L, induced the expansion of DC precursors for >4 wk, as documented by secondary clonogenic assays. This demonstrates that although GM-CSF and TNF-alpha do not require additional cytokines to generate DC from primitive human CD34(+)DR(-) progenitor cells, they do force terminal differentiation of DC precursors. Conversely, FLT3-L and SCF do not directly affect DC differentiation, but instead sustain the long-term expansion of CFU-DC, which can be induced to produce mature DC by GM-CSF and TNF-alpha.
    The Journal of Immunology 02/2001; 166(2):848-54. · 5.52 Impact Factor
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    ABSTRACT: Dendritic cells (DC) are the most powerful antigen presenting cells (APC) and play a pivotal role in initiating the immune response. In light of their unique properties, DC have been proposed as a tool to enhance immunity against infectious agents and in anticancer vaccine strategies. In the last few years, the development of DC has been extensively investigated. The present paper summarizes the most recent findings on the differentiation of myeloid DC from hematopoietic CD34+ progenitors and methods for DC generation in vitro. A better understanding of DC function has important implications for their use in clinical settings.
    Journal of biological regulators and homeostatic agents 01/2001; 15(1):49-52. · 5.18 Impact Factor
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    ABSTRACT: To generate mature and fully functional CD83(+) dendritic cells derived from circulating CD14(+) cells highly purified from the leukapheresis products of multiple myeloma patients.CD14(+) monocytes were selected by high-gradient magnetic separation and differentiated to immature dendritic cells with granulocyte-macrophage colony-stimulating factor and interleukin-4 for 6-7 days and then induced to terminal maturation by the addition of tumor necrosis factor-alpha or stimulation with CD40 ligand. Dendritic cells were characterized by immunophenotyping, evaluation of soluble antigens uptake, cytokine secretion, capacity of stimulating allogeneic T cells, and ability of presenting nominal antigens, including tumor idiotype, to autologous T lymphocytes. Phenotypic analysis showed that 90% +/- 6% of cells recovered after granulocyte-macrophage colony-stimulating factor and interleukin-4 stimulation expressed all surface markers typical of immature dendritic cells and demonstrated a high capacity of uptaking soluble antigens as shown by the FITC-dextran assay. Subsequent exposure to maturation stimuli induced the downregulation of CD1a and upregulation of CD83, HLA-DR, costimulatory molecules and induced the secretion of large amounts of interleukin-12. Mature CD83(+) cells showed a diminished ability of antigen uptake whereas they proved to be potent stimulators of allogeneic T cells in a mixed lymphocyte reaction. Monocyte-derived dendritic cells, pulsed before the addition of maturation stimuli, were capable of presenting soluble proteins such as keyhole limpet hemocyanin and tetanus toxoid to autologous T cells for primary and secondary immune response, respectively. Conversely, pulsing of mature (CD83(+)) dendritic cells was less efficient for the induction of T-cell proliferation. More importantly, CD14(+) cells-derived dendritic cells stimulated autologous T-cell proliferation in response to a tumor antigen such as the patient-specific idiotype. Moreover, idiotype-pulsed dendritic cells induced the secretion of interleukin-2 and gamma-interferon by purified CD4(+) cells. T-cell activation was better achieved when Fab immunoglobulin fragments were used as compared with the whole protein. When dendritic cells derived from CD14(+) cells from healthy volunteers were analyzed, we did not find any difference with samples from myeloma patients as for cell yield, phenotypic profile, and functional characteristics. These studies demonstrate that mobilized purified CD14(+) cells represent the optimal source for the production of a homogeneous cell population of mature CD83(+) dendritic cells suitable for clinical trials in multiple myeloma.
    Experimental Hematology 09/2000; 28(8):931-40. · 2.91 Impact Factor
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    ABSTRACT: Transforming growth factor beta3 (TGF-beta3) is a potent suppressor of human hematopoietic progenitor cells. In this article, we compare the activity of TGF-beta3 on highly purified CD34+ cells and more immature CD34-DR(-) cells from chronic myelogenous leukemia (CML) patients in chronic phase and normal donors. Primitive hematopoietic progenitors were stimulated in liquid cultures and clonogenic assays by early-acting growth factors such as stem cell factor (SCF) and interleukin 11 (IL-11) and the intermediate-late-acting stimulating factors IL-3, granulocyte-macrophage colony-stimulating factor, and erythropoietin. Molecular analysis of bcr/abl mRNA was performed on single CML colonies by nested reverse transcriptase polymerase chain reaction. Moreover, cell cycle analysis and assessment of apoptosis of normal and leukemic CD34+ cells were performed by propidium iodide (PI) alone and simultaneous staining with annexin V and PI, respectively. The colony-forming efficiency of CML CD34+ cells was generally inhibited by more than 90% regardless of whether the colony-stimulating factors were used alone or combined. When compared to normal CD34+ cells, leukemic cells were significantly more suppressed in 6 of 8 culture conditions. The inhibitory effect of TGF-beta3 on CD34+ cells was exerted within the first 24 hours of incubation as demonstrated by short-term preincubation followed by IL-3-and SCF-stimulated colony assays. Evaluation of bcr/abl transcript on residual CML colonies incubated with TGF-beta3 demonstrated a small subset of neoplastic CD34+ cells unresponsive to the inhibitory effect of the study cytokine. TGF-beta3 demonstrated a greater inhibitory activity on primitive CD34+DR cells than on more mature CD34+ cells. Again, CML CD34+DR(-) cells were significantly more inhibited by TGF-beta3 than their normal counterparts in 3 of 8 culture conditions. Kinetic analysis performed on CD34+ cells showed that TGF-beta induces cell cycle arrest in G(1) phase. However, this mechanism of action is shared by normal and leukemic cells. Conversely, TGF-beta3 preferentially triggered the programmed cell death of CML CD34-cells without increasing the proportion of leukemic cells coexpressing CD95 (Fas receptor), and this effect was not reversed by functional blockade of Fas receptor. Conclusion. We demonstrate that TGF-beta3 exerts a potent suppressive effect on CML cells that is partly mediated by Fas-independent apoptosis.
    Experimental Hematology 08/2000; 28(7):775-83. · 2.91 Impact Factor
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    ABSTRACT: Objective Transforming growth factor β3 (TGF-β3) is a potent suppressor of human hematopoietic progenitor cells. In this article, we compare the activity of TGF-β3 on highly purified CD34+ cells and more immature CD34+DR− cells from chronic myelogenous leukemia (CML) patients in chronic phase and normal donors.
    Experimental Hematology - EXP HEMATOL. 01/2000; 28(7):775-783.
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    ABSTRACT: CD40 antigen is a costimulatory molecule highly expressed on dendritic cells (DC) and activated B cells, which induces T-cell proliferation through the binding with CD40L receptor. In this study, we evaluated CD40 expression on normal CD34(+) blood cells and functionally characterized CD34(+)CD40(+) and CD34(+)CD40(-) cell subsets. CD40, CD80, and CD86 antigens were constitutively expressed on 3.2% +/- 4.5%, 0%, and 1.8% +/- 1.2% CD34(+) blood cells, respectively. However, after 24 hours in liquid culture with medium alone, or with tumor-necrosis-factor-alpha (TNF-alpha), or with allogeneic mononuclear cells 10.8% +/- 3.8%, 75.3% +/- 15.0% and 53. 7% +/- 17.0% CD34(+) blood cells, respectively, became CD40(+). After incubation for 24 hours with TNF-alpha CD34(+)CD40(+) blood cells expressed only myeloid markers and contained less than 5% CD86(+) and CD80(+) cells. Also, a 24-hour priming with TNF-alpha or ligation of CD40 significantly increased the CD34(+) blood cells alloantigen presenting function. Finally, purified CD34(+)CD40(+) blood cells stimulated an alloreactive T-cell response in MLC, were enriched in granulocytic, monocytic, and dendritic precursors, and generated high numbers of DC in 11-14 d liquid cultures with GM-CSF, SCF, TNF-alpha and FLT-3L. In contrast, CD34(+)CD40(-) cells were poorly immunogenic, contained committed granulocytic and erythroid precursors and early progenitors, and differentiated poorly toward the DC lineage. In conclusion, a short incubation with TNF-alpha allows the selection of CD40(+) blood progenitors, which may be a useful source of DC precursors for antitumor vaccine studies, and also a CD34(+)CD40(-) blood cell fraction that could be exploited in innovative strategies of allogeneic transplantation across HLA barriers.
    Blood 11/1999; 94(7):2293-300. · 9.78 Impact Factor
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    ABSTRACT: We review here the functional and kinetic characteristics of highly purified hematopoietic CD34+ mobilized into peripheral blood (PB) by granulocyte colony-stimulating factor (G-CSF) with or without chemotherapy for autologous or allogeneic transplantation. Circulating CD34+ cells were evaluated for their colony-forming capacity and trilineage proliferative response to selected recombinant human (rh) CSF in vitro, and the content of very primitive long-term culture initiating cells (LTC-IC). In addition, the cycling status of PB CD34+ cells, including committed clonogenic progenitor cells and the more immature LTC-IC, was determined by the cytosine arabinoside (Ara-C) suicide test and the acridine orange (AO) flow cytometric technique. By comparison, bone marrow (BM) CD34+ cells from the same individuals were studied under steady-state conditions and during G-CSF administration. Clonogenic assays in methylcellulose showed the same frequency of colony-forming unit cells (CFU-C) when PB primed-CD34+ cells and BM cells were stimulated with phytohemagglutinin-lymphocyte-conditioned medium (PHA-LCM). However, mobilized CD34+ cells were significantly more responsive than their steady-state BM counterparts to interleukin-3 (IL-3) and stem cell factor (SCF) combined with G-CSF or IL-3 in the presence of erythropoietin (Epo). Conversely, circulating and BM megakaryocyte precursors (CFU-MK) showed the same clonogenic efficiency in response to IL-3, GM-CSF and IL-3, IL-6 and Epo. Interestingly, very few CD34+ cells expressed the Mpl receptor and this finding resulted in the lower proliferative response of mobilized CFU-MK to the Mpl-ligand (megakaryocyte growth and development factor; MGDF), as compared to BM cells. After 5 weeks of liquid culture supported by the engineered murine stromal cell line M2-10B4 to produce G-CSF and IL-3, we reported a similar frequency of LTC-IC in PB and steady-state BM. Kinetic studies on PB and BM CD34+ cells, including LTC-IC, showed the low number of circulating progenitor cells in S and G2M phase whereas simultaneous DNA/RNA analysis and the Ara-C suicide assay demonstrated that the majority of PB CD34+ cells and LTC-IC are not quiescent (ie in G0 phase) being in G1 phase. Moreover, G-CSF administration prevented apoptosis in a small but significant proportion of mobilized CD34+ cells. Thus, our results indicate that mobilized and BM CD34+ cells can be considered equivalent for the frequency of both committed and more immature hematopoietic progenitor cells, although they show different kinetic and functional profiles. A further set of experiments indicated that G-CSF treatment did not alter the alloantigen presenting function of CD34+ cells which was mainly mediated by the upregulation of costimulatory molecules upon coincubation with allogeneic T cells. Taken together, these findings should allow a better understanding of PBSC transplantation.
    Bone Marrow Transplantation 01/1999; 22 Suppl 5:S47-50. · 3.54 Impact Factor
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    ABSTRACT: Dendritic cells (DCs) are the most powerful professional antigen-presenting cells (APC), specializing in capturing antigens and stimulating T-cell-dependent immunity. In this study we report the generation and characterization of functional DCs derived from both steady-state bone marrow (BM) and circulating haemopoietic CD34+ cells from 14 individuals undergoing granulocyte colony-stimulating factor (G-CSF) treatment for peripheral blood stem cells (PBSC) mobilization and transplantation. Clonogenic assays in methylcellulose showed an increased frequency and proliferation of colony-forming unit-dendritic cells (CFU-DC) in circulating CD34+ cells, compared to that of BM CD34+ precursors in response to GM-CSF and TNF-alpha with or without SCF and FLT-3L. Moreover, peripheral blood (PB) CD34+ cells generated a significantly higher number of fully functional DCs, as determined by conventional mixed lymphocyte reactions (MLR), than their BM counterparts upon different culture conditions. DCs derived from mobilized stem cells were also capable of processing and presenting soluble antigens to autologous T cells for both primary and secondary immune response. Replacement of the early-acting growth factors SCF and FLT-3L with IL-4 at day 7 of culture of PB CD34+ cells enhanced both the percentage of total CD1a+ cells and CD1a+ CD14- cells and the yield of DCs after 14 d of incubation. In addition, the alloreactivity of IL-4-stimulated DCs was significantly higher than those generated in the absence of IL-4. Furthermore, autologous serum collected during G-CSF treatment was more efficient than fetal calf serum (FCS) or two different serum-free media for large-scale production of DCs. Thus, our comparative studies indicate that G-CSF mobilizes CD34+ DC precursors into PB and circulating CD34+ cells represent the optimal source for the massive generation of DCs. The sequential use of early-acting and intermediatelate-acting colony-stimulating factors (CSFs) as well as the use of autologous serum greatly enhanced the growth of DCs. These data may provide new insights for manipulating immunocompetent cells for cancer therapy.
    British Journal of Haematology 07/1998; 101(4):756-65. · 4.94 Impact Factor
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    ABSTRACT: In this study, the hypothesis that a subset of granulocyte colony-stimulating factor (G-CSF)-mobilized CD34+ blood cells may actively induce an allogeneic T cell response in vitro was tested. Circulating CD34+ cells were purified to > or =98% by high gradient magnetic separation and then analyzed for the coexpression of HLA-DR, the common beta-chain of the leukointegrin family CD18 and costimulatory molecules CD80 (B7-1) and CD86 (B7-2). These antigens were expressed on average on: 94.9 +/- 2.5%, 64.4 +/- 15.4%, 0% and 1.9 +/- 1.2% CD34+ blood cells, respectively. Irradiated CD34+ cells induced a high proliferative response of allogeneic, but not autologous, purified CD4+ and CD8+ T cells in primary mixed leukocyte culture (MLC). An average three-fold lower CD4+ and CD8+ T cell response was induced by mononuclear cells from G-CSF-treated donors. A lower frequency of allostimulating cells among mononuclear cells rather than among CD34+ cells in the apheresis was documented by limiting dilution assay (LDA). As previously observed with marrow, sorted CD34+/CD18+ cells induced the proliferation of allogeneic T cells in MLC, while CD34+/CD18- cells, which were >94% HLA-DR+ and contained both committed (CFU-C) and early (LTC-IC) hematopoietic progenitors, stimulated allogeneic T cells poorly. Three-color staining cytofluorimetry indicated that expression of CD80 and CD86 were upregulated in 6.9 +/- 4.9 and 10.7 +/- 2.6% CD34+ blood cells respectively, after 24-30 h of culture with autologous or allogeneic mononuclear cells, or with CD4+, or CD8+ T cells, but not with medium alone. Moreover, the upregulation of CD86 was observed on CD34+/CD18+ rather than on CD34+/CD18- cells after 30 h in MLC. Blocking experiments demonstrated that preincubation of stimulator and responder cells with anti-CD80 plus anti-CD86 monoclonal antibodies induced a 84 +/- 8% inhibition of CD34+ cell allostimulating activity after 6 days in primary MLC. These results suggest that G-CSF-mobilized CD34+ hematopoietic progenitors with alloantigen presenting function express CD18 and may upregulate CD80 and CD86 upon interaction with T cells. Since activation of B7 costimulatory molecules represents an active costimulatory pathway on G-CSF-mobilized CD34+ cells, the blockade of these molecules or, alternatively, the use of selected non-immunogenic CD34+/CD18- blood stem cells may represent a new strategy for reducing graft rejection and overcoming HLA barriers in allogeneic stem cell transplantation.
    Bone Marrow Transplantation 06/1998; 21(12):1183-91. · 3.54 Impact Factor
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    ABSTRACT: Dendritic cells (DCs) are the most powerful professional antigen-presenting cells (APC), specializing in capturing antigens and stimulating T-cell-dependent immunity. In this study we report the generation and characterization of functional DCs derived from both steady-state bone marrow (BM) and circulating haemopoietic CD34+ cells from 14 individuals undergoing granulocyte colony-stimulating factor (G-CSF) treatment for peripheral blood stem cells (PBSC) mobilization and transplantation. Clonogenic assays in methylcellulose showed an increased frequency and proliferation of colony-forming unit-dendritic cells (CFU-DC) in circulating CD34+ cells, compared to that of BM CD34+ precursors in response to GM-CSF and TNF-α with or without SCF and FLT-3L. Moreover, peripheral blood (PB) CD34+ cells generated a significantly higher number of fully functional DCs, as determined by conventional mixed lymphocyte reactions (MLR), than their BM counterparts upon different culture conditions. DCs derived from mobilized stem cells were also capable of processing and presenting soluble antigens to autologous T cells for both primary and secondary immune response. Replacement of the early-acting growth factors SCF and FLT-3L with IL-4 at day 7 of culture of PB CD34+ cells enhanced both the percentage of total CD1a+ cells and CD1a+CD14− cells and the yield of DCs after 14 d of incubation. In addition, the alloreactivity of IL-4-stimulated DCs was significantly higher than those generated in the absence of IL-4. Furthermore, autologous serum collected during G-CSF treatment was more efficient than fetal calf serum (FCS) or two different serum-free media for large-scale production of DCs. Thus, our comparative studies indicate that G-CSF mobilizes CD34+ DC precursors into PB and circulating CD34+ cells represent the optimal source for the massive generation of DCs. The sequential use of early-acting and intermediate-late-acting colony-stimulating factors (CSFs) as well as the use of autologous serum greatly enhanced the growth of DCs. These data may provide new insights for manipulating immunocompetent cells for cancer therapy.
    British Journal of Haematology 05/1998; 101(4):756 - 765. · 4.94 Impact Factor
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    ABSTRACT: CD34+ and CD34+ DR- cells from the bone marrow (BM) of chronic-phase chronic myelogenous leukaemia (CML) patients at diagnosis were tested for their colony-forming ability in response to early and intermediate-late colony stimulating factors (CSFs). Molecular analysis revealed that 55.6+/-9% SD of CD 34+ DR- colonies, in which actin and ABL mRNA were detectable, expressed the product of the BCR-ABL gene. The percentage and the clonogenic efficiency of CML DR- cells were significantly lower than those of comparable DR- cells from normal donors. However, clonogenic assays using recombinant human CSFs demonstrated a remarkable proliferation of CML cells when stimulated by SCF, IL-11 and IL-3, used as single factors in the presence of erythropoietin (EPO) and was almost entirely due to erythroid progenitors. Conversely, optimal stimulation of CD34 +DR- cells from normal donors required co-incubation with three or more CSFs. Stroma-noncontact long-term cultures were then established in the presence of exogenous CSFs and human irradiated allogeneic stromal layers or the murine stromal cell line M2-10B4, engineered to produce G-CSF and IL-3. In these cultures the combination of SCF and IL-3 induced a 25.4 +/- 5 SD, 40 +/- 6 SD and 20.5 +/- 6 SD fold increase of colony-forming unit cells (CFU-C), at weeks 2, 4 and 5, respectively. At the same time-points the number of primitive long-term culture initiating cells (LTC-IC) showed a 4 +/- 2 SD, 3.3 +/- 1.5 SD and 2.3 +/-1 SD fold increase compared to baseline values. BCR-ABL mRNA analysis of single colonies demonstrated that 27 +/- 9% SD and 7 +/- 3% SD CFU-C at weeks 4 and 5, respectively, expressed the fusion gene, whereas leukaemic LTC-IC disappeared from the culture by week 2. These results suggest that leukaemic CD34+ DR- cells have a different pattern of response to CSFs than normal cells. In addition, we established culture conditions which allow selective expansion of benign haemopoietic cells coexisting with leukaemic progenitors.
    British Journal of Haematology 05/1998; 101(1):119-29. · 4.94 Impact Factor
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    ABSTRACT: Here we review our recent experience addressing the issue of positive selection and transplantation of hematopoietic CD34+ cells to reduce neoplastic contamination in peripheral blood (PB) autografts from patients with multiple myeloma (MM). We evaluated PB samples from 30 pretreated MM patients following the administration of high dose cyclophosphamide (Cy; 7g/m2 or 4g/m2) and granulocyte-colony stimulating factor (G-CSF), for collection of circulating stem cells (PBSC) to support hematopoietic reconstitution following myeloablative radio-chemotherapy. Twenty six patients showed adequate mobilization of CD34+ progenitor cells and were submitted to PBSC collection. Circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, cryopreserved, and used to reconstitute BM function after myeloablative therapy in 13 patients. The median purity of the enriched CD34+ cell population was 89.5% (range 51-94%) with a 75-fold increase compared to the pretreatment samples. The median overall recovery of CD34+ cells and CFU-GM was 58% (range 33-95%) and 45% (range 7-100%), respectively. Positive selection of CD34+ cells resulted in 2.5-3 log of plasma cells and CD19+ B-lineage cells depletion as determined by immunofluorescence studies, although DNA analysis of CDR III region of IgH gene demonstrated the persistence of minimal residual disease (MRD) in 5 out of 6 patient samples studied. Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (TBI, 1000 cGy) and high dose Melphalan (140 mg/m2) or Melphalan (200 mg/m2) alone. They received a median of 5 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis: the median time to 0.5 x 10(9) neutrophils, 20 and 50 x 10(9) platelets/L of PB was 10, 11 and 12 days, respectively. When we analyzed the immunological reconstitution of this group of patients, we observed a rapid and full recovery of total lymphocyte and NK cell count, although the absolute CD4+ cell count was lower than pretreatment level. These results, as well as other clinically significant parameters, did not significantly differ from those of patients (=13) receiving unmanipulated PBSC following the same pretransplant conditioning regimen. The results of this trial demonstrate that positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3 log and provides a cell suspension capable of restoring a normal hematopoiesis after a TBI-containing conditioning regimen. Based on this pilot trial, we have recently started a clinical study involving a double autotransplant, conditioned with melphalan (200 mg/m2) followed by melphalan (140 mg/m2) and busulphan (14 mg/kg), supported by the reinfusion of highly purified CD34+ cells.
    Leukemia and Lymphoma 01/1998; 26 Suppl 1:1-11. · 2.61 Impact Factor
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    ABSTRACT: In this paper we describe an experimental model for ex vivo purging of contaminating tumor cells from peripheral blood stem cell (PBSC) collections obtained from patients with acute myeloblastic leukemia (AML). We studied the combination of the alkylating agent nitrogen mustard (NM; concentrations ranging from 0.25 to 1.25 microg/mL) and etoposide (VP-16; constant dose of 20 microg/mL), and the conventional cyclophosphamide (Cy)-derivative mafosfamide (concentrations: 20-175 microg/mL). The aims of our study were: 1) To compare the toxicity of the purging protocols on bone marrow (BM) and circulating trilineage precursors collected from normal donors after priming with granulocyte colony-stimulating factor (G-CSF) or after complete remission (CR) consolidation chemotherapy and G-CSF (leukemic patients); 2) to demonstrate the survival of very primitive hematopoietic progenitors (LTC-IC) in the peripheral blood (PB) and the BM after pharmacological treatment; and 3) to evaluate the antineoplastic efficacy of purging protocols on PBSC collections using 3 well-established leukemic cell lines. Our results demonstrated that the toxicity on BM and PB progenitor cells could be correlated with the complete killing of committed granulocyte-macrophage colony-forming units (CFU-GMs) and erythroid precursors (BFU-Es), a condition reached at the concentration of 1.5 microg/mL of NM (in addition to 20 microg/mL of VP-16) and 175 microg/mL of mafosfamide. Notably, early and late megakaryocyte progenitor cells (CFU-MKs and BFU-MKs, respectively) showed higher sensitivity to NM/VP-16, but not to mafosfamide, than did CFU-GMs and BFU-Es. The dose of NM capable of inhibiting 95% of CFU-MKs and BFU-MKs (ID95) was 0.75 microg/mL. After incubation with the same dose of NM, the recovery of CFU-GMs and BFU-Es was 20 +/- 8% SD and 25 +/- 10% SD, respectively (p < 0.05). Long-term liquid cultures showed the recovery of primitive hematopoietic cells after incubation with the highest concentrations of NM/VP-16 and mafosfamide, with no significant differences between PB and BM samples. Under the same experimental conditions, we observed a more than 5-log reduction of contaminating leukemic cell lines (i.e., K-562, KG-1, and HL-60). In conclusion, we demonstrated that NM/VP-16 and mafosfamide purging agents are capable of killing leukemic cell lines that contaminate leukapheresis products from patients with AML, whereas an acceptable proportion of primitive LTC-IC is spared. Moreover, despite the different kinetic and functional profile of mobilized and steady-state BM progenitors, we did not observe any difference in toxicity of antineoplastic agents on hematopoietic cells at different levels of differentiation. These data suggest that pharmacological strategies developed for eliminating minimal residual disease (MRD) from BM autografts can be effectively and safely applied to circulating stem cell harvests.
    Experimental Hematology 12/1997; 25(12):1261-9. · 2.91 Impact Factor
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    ABSTRACT: To clarify the bioenergetic relevance of mtDNA mutations in Leber's hereditary optic neuropathy (LHON), we investigated affected individuals and healthy carriers from six Italian LHON families harboring the 11778/ND4 and the 3460/ND1 mtDNA mutations. The enzymatic activities of mitochondrial complex I and its sensitivity to the potent inhibitors rotenone and rolliniastatin-2 were studied in mitochondrial particles from platelets, in correlation with mtDNA analysis of platelets and leukocytes. In platelets homoplasmic for mutant mtDNA, both 11778/ND4 and 3460/ND1 mutations induced resistance to rotenone and the 3460/ND1 mutation also provoked a marked decrease in the specific activity of complex I. Individuals heteroplasmic in platelets for either mutation showed normal biochemical features, indicating functional complementation of wild-type mtDNA. There was no correlation between the clinical status and mtDNA homo/heteroplasmy in platelets, but the biochemical features correlated with the mitochondrial genotype of platelets. In some cases, the degree of mtDNA heteroplasmy differed in platelets and leukocytes from the same individual with a prevalence of wild-type mtDNA in the platelets. These results imply that biochemical studies on mitochondrial diseases should always be integrated with mtDNA analysis of the same tissue investigated and also suggest that the mtDNA analysis on the leukocyte fraction, as usually performed in LHON, does not necessarily reflect the mutant genotype level of other tissues. The differential tissue heteroplasmy may be more relevant than previously thought in determining disease penetrance.
    Neurology 07/1997; 48(6):1623-32. · 8.30 Impact Factor