Dendritic cell differentiation.
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Cell Differentiation↗
Biomedical subjects
Antonio Curti
Publications and source records attributed to Antonio Curti.
Granulocyte colony-stimulating factor promotes the generation of regulatory DC through induction of IL-10 and IFN-alpha.
We have recently demonstrated that G-CSF promotes the generation of human T regulatory (T(REG)) type 1 cells. In this study, we investigated whether the immunomodulatory effects of G-CSF might be mediated by DC. CD14(+) monocytes were cultured with serum collected after clinical administration of G-CSF (post-G), which contained high amounts of IL-10 and IFN-alpha. Similar to incompletely matured DC, monocytes nurtured with post-G serum acquired a DC-like morphology, expressed high levels of costimulatory molecules and HLA-DR, and exhibited diminished IL-12p70 release and poor allostimulatory capacity. Importantly, post-G DC-like cells were insensitive to maturation stimuli. As shown by neutralization studies, IFN-alpha and, even more pronounced, IL-10 contained in post-G serum inhibited IL-12p70 release by post-G DC-like cells. Furthermore, phenotypic and functional features of post-G DC-like cells were replicated by culturing post-G monocytes with exogenous IL-10 and IFN-alpha. Post-G DC-like cells promoted Ag-specific hyporesponsiveness in naive allogeneic CD4(+) T cells and orchestrated a T(REG) response that was dependent on secreted TGF-beta 1 and IL-10. Finally, neutralization of IL-10 and IFN-alpha contained in post-G serum translated into abrogation of the regulatory features of post-G DC-like cells. This novel mechanism of immune regulation effected by G-CSF might be therapeutically exploited for tolerance induction in autoimmune disorders.
Adult↗
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.
Cancer Vaccines↗
Autologous transplantation of granulocyte colony-stimulating factor-primed bone marrow is effective in supporting myeloablative chemotherapy in patients with hematologic malignancies and poor peripheral blood stem cell mobilization.
We assessed the hematopoietic recovery and transplantation-related mortality (TRM) of patients who had failed peripheral blood stem cell mobilization and subsequently received high-dose chemotherapy supported by granulocyte colony-stimulating factor (G-CSF)-primed bone marrow (BM). Studied were 86 heavily pretreated consecutive patients with acute leukemia (n = 21), refractory/relapsed non-Hodgkin lymphoma (n = 41) and Hodgkin disease (n = 17), and multiple myeloma (n = 7). There were 78 patients who showed insufficient mobilization of CD34+ cells (< 10 cells/microL), whereas 8 patients collected less than 1 x 106 CD34+ cells/kg. BM was primed in vivo for 3 days with 15 to 16 microg/kg of subcutaneous G-CSF. Median numbers of nucleated cells, colony-forming unit cells (CFU-Cs), and CD34+ cells per kilogram harvested were 3.5 x 10(8), 3.72 x 10(4), and 0.82 x 10(6), respectively. Following myeloablative chemotherapy, median times to achieve a granulocyte count higher than 0.5 x 10(9)/L and an unsupported platelet count higher than 20 and 50 x 10(9)/L were 13 (range, 8-24), 15 (range, 12-75), and 22 (range, 12-180) days, respectively, for lymphoma/myeloma patients and 23 (range, 13-53), 52 (range, 40-120), and 90 (range, 46-207) days, respectively, for leukemia patients. Median times to hospital discharge after transplantation were 17 (range, 12-40) and 27 (range, 14-39) days for lymphoma/myeloma and acute leukemia patients, respectively. TRM was 4.6%, whereas 15 patients died of disease. G-CSF-primed BM induces effective multilineage hematopoietic recovery after high-dose chemotherapy and can be safely used in patients with poor stem cell mobilization.
Adolescent↗
Generation of dendritic cells from CD14+ monocytes positively selected by immunomagnetic adsorption for multiple myeloma patients enrolled in a clinical trial of anti-idiotype vaccination.
Circulating monocytes from multiple myeloma patients enrolled in a clinical study of anti-idiotype vaccination were labelled with clinical-grade anti-CD14 microbeads and positively selected with the CliniMACS instrument. Cells were then grown, according to good manufacturing practice guidelines, in fetal-calf-serum-free medium in cell culture bags and differentiated to dendritic cells (DC) with granulocyte-macrophage colony stimulating factor plus interleukin 4 (IL-4), followed by either tumour necrosis factor-alpha (TNF-alpha) or a cocktail of IL-1beta, IL-6, TNF-alpha and prostaglandin-E2. The CD14+ cell yield was increased from 17.6 +/- 6.5% to 93.8 +/- 6.3% (recovery 64.4 +/- 15.4%, viability > 97%). After cell culture, phenotypic analysis showed that 86.7 +/- 6.8% of the cells were DC: 2.27 +/- 0.9 x 108 DC/leukapheresis were obtained, which represented 20.7 +/- 4.6% of the initial number of CD14+ cells. Notably, the cytokine cocktail induced a significantly higher percentage and yield (28.6 +/- 3% of initial CD14+ cells) of DC than TNF-alpha alone, with secretion of larger amounts of IL-12, potent stimulatory activity on allogeneic T cells and efficient presentation of tumour idiotype to autologous T cells. Storage in liquid nitrogen did not modify the phenotype or functional characteristics of preloaded DC. The recovery of thawed, viable DC was 78 +/- 10%. Finally, interferon-alpha-2b was at least as efficient as IL-4 in inducing the differentiation of mature, functional DC from monocytes.
Antibodies, Anti-Idiotypic↗
Functional and kinetic characterization of granulocyte colony-stimulating factor-primed CD34- human stem cells.
We assessed the functional properties and the kinetic status in vitro, and the engraftment potential in vivo of human haematopoietic stem cells according to the expression of CD34 antigen. Lin-CD34- and Lin-CD34+ cells were isolated from granulocyte colony-stimulating factor-primed peripheral blood (PB) cells of healthy donors. The CD34- cell fraction did not contain either clonogenic cells in semisolid culture or long-term culture initiating cells (LTC-IC). However, stroma-dependent liquid cultures and cytokines induced CD34 expression on a minority of stem cells, acquisition of clonogenic capacity and generation of LTC-IC. Significantly higher percentages of quiescent G0 cells and lower percentages of cycling G1 cells were found in Lin-CD34- cells when compared with Lin-CD34+ cells. Kinetic quiescence of Lin-CD34- cells was associated with a significantly higher expression of the negative regulators of the cell cycle, p27Kip1 and p21(cip1/waf1). Cytokine-mediated induction of CD34, in vitro, resulted in cycling of stem cells and downregulation of p27. There was a higher rate of human long-term engraftment in immunocompromised non-obese diabetic (NOD)/recombination activating gene 1null and NOD/severe combined immunodeficient-beta2microglobulin(null) mice injected with CD34+ cells. Thus, our study indicated that CD34 expression on human PB stem cells was associated with haematopoietic activity, cell-cycle recruitment and downregulation of p27Kip1 in vitro and higher engraftment capacity in vivo.
Animals↗
Autologous and MHC class I-negative allogeneic tumor cells secreting IL-12 together cure disseminated A20 lymphoma.
Cytokine gene-modified tumor cells have increased immunogenicity and retain the antigenic repertoire of a particular neoplasia. However, practical concerns have led to an increased interest in allogeneic gene-transduced bystander cells as a broader source of cytokines for autologous tumor cell-based vaccines. Here, we show that allogeneic B78H1 major histocompatibility complex (MHC) class I-negative and -positive (H-2K(b)- and D(b)-transfected) cells induced cytotoxic T lymphocytes (CTLs) and protection in BALB/c mice at comparable levels in response to a challenge with C26 (H-2(d)) colon carcinoma cells sharing the tumor-associated antigen envelope glycoprotein 70 (env-gp70) with both cell lines. Class I-negative B78H1 cells transduced to express interleukin-12 (IL-12) and mixed with autologous A20 tumor cells led to eradication of preestablished A20 lymphoma in 50% or 100% of treated mice after 3 or 4 vaccinations, respectively, whereas A20 cells alone or mixed with nontransduced B78H1 cured none or 50% of mice after 3 or 4 vaccinations, respectively. Immunization with the IL-12-producing bystander cell line increased tumor-specific proliferation and type 1 cytokine production by CD4(+) T cells. By contrast, CD4 T-cell function appeared impaired after immunization with A20 cells alone or mixed with B78H1 cells. Indeed, only CD4(+) T cells from IL-12-treated mice could be restimulated with anti-OX40 monoclonal antibody (mAb) in place of a fourth cellular boost. Moreover, the IL-12-based tumor vaccine induced expansion of tumor-specific interferon-gamma (IFN-gamma)-producing CD8(+) T cells. These results are clinically relevant for the development of feasible IL-12 cancer vaccines based on engineered class I-negative bystander cells.
Animals↗
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.
Antigen Presentation↗
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.
Apoptosis↗
The therapeutic role of dendritic cells in cancer immunotherapy.
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Animals↗