Activity of lipoplatin in tumor and in normal cells in vitro.

Lipoplatin is a novel liposomal cisplatin formulation with reduced adverse side effects compared with its parental compound, cisplatin. The aims of this preclinical study were to compare lipoplatin and cisplatin cytotoxicity in vitro in established cell lines derived from non-small cell lung cancer, renal cell carcinoma, and in normal hematopoietic cell precursors, and to identify biological markers associated with sensitivity and resistance. Our results showed a superior cytotoxicity in all tumor cell models and a much lower toxicity in normal cells for lipoplatin compared with cisplatin, suggesting a higher therapeutic index for the liposomal compound. Moreover, RT-PCR analysis of molecular markers known to be related to cisplatin resistance showed a direct correlation between cisplatin and lipoplatin resistance and ERCC1 and LRP expression. In conclusion, lipoplatin showed a higher antitumor activity in both tumor histotypes investigated and was found to be safer than the parent compound, cisplatin. Moreover, ERCC1 and LRP expression levels would seem to be valid predictors of sensitivity or resistance to these drugs.

Generation of dendritic cells from positively selected CD14+ monocytes for anti-tumor immunotherapy.

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.

CTLA-4 is not restricted to the lymphoid cell lineage and can function as a target molecule for apoptosis induction of leukemic cells.

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.

Dendritic cells are functionally defective in multiple myeloma: the role of interleukin-6.

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.

Interleukin-11 induces proliferation of human T-cells and its activity is associated with downregulation of p27(kip1).

BACKGROUND AND OBJECTIVES: 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. DESIGN AND METHODS: 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. RESULTS: 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. INTERPRETATION AND CONCLUSIONS: 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.