Andrea Berer

1,25-Dihydroxyvitamin D3 inhibits dendritic cell differentiation and maturation in vitro

OBJECTIVE Because of its potent immunosuppressive properties in vitro as well as in vivo, we studied the effect of 1,25-dihydroxyvitamin D(3) (calcitriol) on differentiation, maturation, and function of dendritic cells (DC). MATERIALS AND METHODS Monocyte-derived DCs were generated with GM-CSF plus IL-4, and maturation was induced by a 2-day exposure to TNFalpha. DCs were derived from CD34(+) progenitors using SCF plus GM-CSF plus TNFalpha. For differentiation studies, cells were exposed to calcitriol at concentrations of 10(-)(9)- 10(-7) M at days 0, 6, and 8, respectively. The obtained cell populations were evaluated by morphology, phenotype, and function. RESULTS When added at day 0, calcitriol blocked DC differentiation from monocytes and inhibited the generation of CD1a(+) cells from progenitor cells while increasing CD14(+) cells. Exposure of immature DCs to calcitriol at day 6 resulted in a loss of the DC-characteristic surface molecule CD1a, downregulation of the costimulatory molecules CD40 and CD80, and MHC class II expression, whereas the monocyte/macrophage marker CD14 was clearly reinduced. In addition, calcitriol hindered TNFalpha-induced DC maturation, which is usually accompanied with induction of CD83 expression and upregulation of costimulatory molecules. In contrast, the mature CD83(+) DCs remained CD1a(+)CD14(-) when exposed to calcitriol. The capacity of cytokine-treated cells to stimulate allogeneic and autologous T cells and to take up soluble antigen was inhibited by calcitriol. CONCLUSION The potent suppression of DC differentiation, the reversal of DC phenotype, and function in immature DCs, as well as the inhibition of DC maturation by calcitriol, may explain some of its immunosuppressive properties.

Calcium ionophore: a single reagent for the differentiation of primary human acute myelogenous leukaemia cells towards dendritic cells.

Blood monocytes and CD34+ haemopoietic progenitor cells, as well as certain leukaemic cell lines, acquire characteristics of mature dendritic cells (DC) after stimulation with calcium ionophore (CI). We studied whether the in vitro treatment of primary human acute myelogenous leukaemia (AML) cells with CI leads to differentiation towards DC. Blast cells derived from nine AML patients were cultured in the presence of either CI or an established differentiation cocktail consisting of granulocyte‐macrophage colony‐stimulating factor plus interleukin 4 and tumour necrosis factor‐α for 5–7 d. Microscopic examination revealed that under both conditions, AML cells were shifted along the DC pathway. In seven out of nine cases, CI‐cultivation led to a higher proportion of cells with dendritic morphology. The percentage of CD40 and CD86 expressing cells was significantly increased upon CI treatment compared with cytokine‐cultured cells. DC molecules as CD80 and CD83 were up‐regulated upon calcium mobilization of AML cells in four out of nine samples. In four cases, CI‐treated stimulator cells induced an enhanced proliferative allogeneic T‐cell response compared with cytokine‐treated stimulator cells. In conclusion, these data demonstrate that CI treatment is an alternative in vitro strategy to differentiate human AML cells into DC.

Interleukin-10 inhibits burst-forming unit-erythroid growth by suppression of endogenous granulocyte-macrophage colony-stimulating factor production from T cells

Numerous cytokines released from accessory cells have been shown to exert either stimulatory or inhibitory growth signals on burst-forming unit-erythroid (BFU-E) growth. Because of its cytokine synthesis-inhibiting effects on T cells and monocytes, interleukin-10 (IL-10) may be a potential candidate for indirectly affecting erythropoiesis. We investigated the effects of IL-10 on BFU-E growth from normal human peripheral blood mononuclear cells (PBMC) using a clonogenic progenitor cell assay. The addition of recombinant human IL-10 to cultures containing recombinant human erythropoietin suppressed BFU-E growth in a dose-dependent manner (by 55.2%, range 47.3-63.3%, p < 0.01, at 10 ng/mL). In contrast, no inhibitory effect of IL-10 was seen when cultivating highly enriched CD34+ cells. BFU-E growth from PBMC also was markedly suppressed in the presence of a neutralizing anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody (by 48.7%, range 32.9-61.2% inhibition,p < 0.01), but not by neutralizing antibodies against granulocyte colony-stimulating factor and interleukin-3. This suggests a stimulatory role of endogenously released GM-CSF on BFU-E formation. Also, the addition of exogenous GM-CSF completely restored IL-10-induced suppression of BFU-E growth. To determine the cellular source of GM-CSF production, we analyzed GM-CSF levels in suspension cultures containing PBMC that were either depleted of monocytes or T cells. Monocyte-depleted PBMC showed spontaneous production of increasing amounts of GM-CSF on days 3, 5, and 7, respectively, which could be suppressed by IL-10, whereas GM-CSF levels did not increase in cultures containing T-cell-depleted PBMC. Our data indicate that IL-10 inhibits the growth of erythroid progenitor cells in vitro, most likely by suppression of endogenous GM-CSF production from T cells.

Culture requirements for induction of dendritic cell differentiation in acute myeloid leukemia

Abstract Peripheral blood mononuclear cells (PBMCs) from 15 newly diagnosed acute myeloid leukemia (AML) patients were cultured in fetal calf serum-free media supplemented with either granulocyte/macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-4 and tumor necrosis factor α (TNFα), or GM-CSF, stem cell factor (SCF), TNFα and transforming growth factor β (TGFβ) in order to generate leukemia-derived dendritic cells (DCs). Cultured cells were analyzed by flow cytometry with respect to DC-associated surface molecules (CD1a, CD83, CD40, CD80, CD86, HLA-DR) when they showed significant DC morphology in culture (14 cases). After cultivation, neo-expression or upregulation of CD1a antigen was found in 8 samples, CD83 in 2, CD40 in 14, CD80 in 7, and CD86 in 9. Twelve of 14 AMLs, in which DC morphology could be induced upon cultivation, showed upregulation of at least 2 DC-associated molecules. For induction of DC differentiation, GM-CSF, IL-4 plus TNFα was superior in 11 cases, and better results were obtained with GM-CSF, SCF, TNFα plus TGFβ in 3 cases. In 7 of 14 samples tested, a marked increase of the T-cell stimulatory capacity could be demonstrated in the allogeneic mixed lymphocyte reaction. The leukemic origin of in vitro-generated DCs was demonstrated by fluorescence in situ hybridization in a patient with translocation t(15;17). Our results suggest that the use of different culture conditions may extend the number of AML patients in which a differentiation towards the DC lineage can be induced in vitro.

Kell is not restricted to the erythropoietic lineage but is also expressed on myeloid progenitor cells

Anti‐Kell antibodies have been shown to suppress fetal erythropoiesis, but little is known about their effect on myelopoiesis. We analysed the effect of Kell‐related antibodies on granulocyte–macrophage colony‐forming units (CFU‐GM) growth in semisolid medium using peripheral blood mononuclear cells (PBMNCs) from haematologically normal individuals. In addition to its inhibitory effect on erythroid burst‐forming units (BFU‐E) growth, anti‐Kell antibodies significantly reduced CFU‐GM colony formation from Kell‐positive individuals but not from Kell‐negative donors. Moreover, anti‐cellano and anti‐Kpb antibodies also inhibited the growth of CFU‐GM from antigen‐positive MNCs. These data indicate that Kell is not restricted to erythroid blood cells, but is also expressed on myeloid progenitor cells.

Cytogenetic risk groups in acute myeloblastic leukaemia differ greatly in their semi-solid colony growth

We have analysed the results of semi‐solid bone marrow cultures in 296 patients with de novo acute myeloblastic leukaemia (AML) and correlated them with the leukaemic karyotype. A favourable prognostic karyotype was found in 52 patients (group A, 18·3%), an intermediate karyotype in 163 patients (group B, 57·4%), and unfavourable cytogenetics were observed in 69 patients (group C, 24·3%). Median colony growth according to the three risk groups was 2 (range 0–344) in group A, 14·5 (range 0–5000) in group B and 50·0 (0–3000) in group C (A vs. B, P < 0·001; A vs. C, P < 0·001; B vs. C, P < 0·01). Among the patients treated with chemotherapy (n = 257), median colony growth was 10 (range 0–5000) in those who achieved complete remission (CR) compared with 56·5 (range 0–1000) in patients without remission (NR) (P = 0·002). The median colony growth of all patients [13/105 bone marrow mononuclear cells (BMMCs); range 0–5000] significantly discriminated between patients regarding survival (OS 11 vs. 7 months, P = 0·044). However, multiple Cox regression analysis revealed cytogenetic risk groups as the most important predictor for achieving CR, disease‐free and overall survival, with colony growth adding no additional prognostic information. In 64 patients, colony growth was also investigated without the addition of exogenous cytokines. Interestingly, none of the patients with a favourable karyotype exhibited autonomous growth, whereas 50% with an intermediate and 73% of patients with an unfavourable karyotype displayed either partial or full autonomous growth in vitro (P = 0·0004). Our data suggest that the growth potential of the leukaemic clone seems to be critically influenced by the molecular changes emerging from chromosomal abnormalities.

Generation of dendritic cells from human chronic myelomonocytic leukemia cells in fetal calf serum-free medium.

It is generally believed that the immune system plays a role not only in the acquisition of malignant diseases but also in the rejection of microscopic as well as established tumor cells. Failure of the immune system to eliminate tumor cells may be, among other factors, due to an insufficient presentation of tumor antigens. Dendritic cells (DCs), as professional antigen-presenting cells, therefore, may be therapeutically used to initiate or enhance immune responses in patients with malignancies. In this study we demonstrate that peripheral blood cells of patients with chronic myelomonocytic leukemia (CMML) can be induced to acquire DC characteristics. Upon culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4) plus tumor necrosis factor α (TNFα), CMML cells develop DC morphology and acquire the phenotypic characteristics of DCs. When a CD14+ cell population is used for DC generation, a homogeneous differentiation towards the DC lineage occurs similar to that, observed in normal peripheral blood monocytes. CMML-derived DCs are potent stimulators of the allogeneic mixed lymphocyte reaction (MLR) when compared with uncultivated cells. The demonstration of a deletion of the long arm of chromosome 7, del(7)(q22), in 86% of highly enriched CD1a+ cells by fluorescence in situ hybridization (FISH) indicates the leukemic origin of generated DCs. In addition, we present data that generation of CMML-derived DCs is also possible under fetal calf serum-free conditions for a potential clinical use. These DCs may be used as a cellular vaccine to induce anti-tumor immunity in patients with CMML.

Relation of In Vitro Growth Characteristics to Cytogenetics and Treatment Outcome in Acute Myeloid Leukemia: Prognostic Significance in Patients with a Normal Karyotype

We analyzed in vitro growth characteristics of bone marrow mononuclear cells (BMMCs) from 322 patients with acute myeloid leukemia (AML) in relation to cytogenetic abnormalities. Median colony growth was low in each of the cytogenetic changes associated with a favorable outcome. Most karyotypic abnormalities in the intermediate prognosis group were associated with low growth potential, but 11q23 abnormalities exhibited 8 times higher in vitro growth. Cytogenetic changes that included abn(3q) seemed to display the highest colony growth in the unfavorable prognosis group, whereas isolated -7 may have been associated with limited growth potential. In vitro growth behavior was predictive of neither rate of complete remission (CR) nor survival of AML patients within the 3 cytogenetic risk groups. In contrast, colony growth differed significantly in the subgroup of patients with a normal karyotype who achieved remission with induction treatment and those who had no remission (10 versus 81.5/105 BMMCs;P = .015). Significantly more patients with normal cytogenetics and colony growth below the 50th percentile went into CR than did patients with colony growth above the 50th percentile (82.8% versus 71.2%). Only 4 (6.8%) of the patients in the low growth group had no remission, compared with 12 (23.1%) of the patients with higher in vitro growth (P = .031, chi-square test). In conclusion, colony growth may prove useful as a prognostic factor for early treatment failure in AML patients with a normal karyotype.Int J Hematol. 2003;78:241-247.

Generation of Leukemia-Derived Dendritic Cells from Patients with AML

Although AML cells are highly sensitive to cytotoxic agents the majority of AML patients relaps after conventional chemotherapy due to the persistence of minimal residual disease. Leukemia cell derived dendritic cells (DCs) generated by in vitro differentiation of AML cells may be useful for anti-leukemia vaccination strategies.