Elena Alfani

The dominant negative β isoform of the glucocorticoid receptor is uniquely expressed in erythroid cells expanded from polycythemia vera patients

Glucocorticoid receptor (GR) agonists increase erythropoiesis in vivo and in vitro. To clarify the effect of the dominant negative GRβ isoform (unable to bind STAT-5) on erythropoiesis, erythroblast (EB) expansion cultures of mononuclear cells from 18 healthy (nondiseased) donors (NDs) and 16 patients with polycythemia vera (PV) were studied. GRβ was expressed in all PV EBs but only in EBs from 1 ND. The A3669G polymorphism, which stabilizes GRβ mRNA, had greater frequency in PV (55%; n = 22; P = .0028) and myelofibrosis (35%; n = 20) patients than in NDs (9%; n = 22) or patients with essential thrombocythemia (6%; n = 15). Dexamethasone stimulation of ND cultures increased the number of immature EBs characterized by low GATA1 and β-globin expression, but PV cultures generated great numbers of immature EBs with low levels of GATA1 and β-globin irrespective of dexamethasone stimulation. In ND EBs, STAT-5 was not phosphorylated after dexamethasone and erythropoietin treatment and did not form transcriptionally active complexes with GRα, whereas in PV EBs, STAT-5 was constitutively phosphorylated, but the formation of GR/STAT-5 complexes was prevented by expression of GRβ. These data indicate that GRβ expression and the presence of A3669G likely contribute to development of erythrocytosis in PV and provide a potential target for identification of novel therapeutic agents.

Identification of Two New Synthetic Histone Deacetylase Inhibitors That Modulate Globin Gene Expression in Erythroid Cells from Healthy Donors and Patients with Thalassemia

We have identified two new histone deacetylase (HDAC) inhibitors (9 and 24) capable of inducing the expression of γ-globin and/or β-globin promoter-driven reporter genes in a synthetic model of Hb switch. Both compounds also increased, with different mechanisms, the γ/(γ+β) ratio expressed in vitro by normal human erythroblasts. Compound 9 increased the levels of γ-globin mRNA and the γ/(γ+β) ratio (both by 2-fold). Compound 24 increased by 3-fold the level of γ-globin and decreased by 2-fold that of β-globin mRNA, increasing the γ/(γ+β) ratio by 6-fold, and raising (by 50%) the cell HbF content. Both compounds raised the acetylation state of histone H4 in primary cells, an indication that their activity was mediated through HDAC inhibition. Compounds 9 and 24 were also tested as γ/(γ+β) mRNA inducers in erythroblasts obtained from patients with β0 thalassemia. Progenitor cells from patients with β0 thalassemia generated in vitro morphologically normal proerythroblasts that, unlike normal cells, failed to mature in the presence of EPO and expressed low β-globin levels but 10 times higher-than-normal levels of the α hemoglobin-stabilizing protein (AHSP) mRNA. Both compounds ameliorated the impaired in vitro maturation in β0 thalassemic erythroblasts, decreasing AHSP expression to normal levels. In the case of two patients (of five analyzed), the improved erythroblast maturation was associated with detectable increases in the γ/(γ+β) mRNA ratio. The low toxicity exerted by compounds 9 and 24 in all of the assays investigated suggests that these new HDAC inhibitors should be considered for personalized therapy of selected patients with β0 thalassemia.

Expression of signal transduction proteins during the differentiation of primary human erythroblasts

The high number (>108–10) of primary human pro‐erythroblasts (CD36high/CD235alow) obtainable in HEMA culture (Migliaccio et al., 2002 ) is exploited here to analyse the expression of proteins implicated in erythropoietin (EPO)‐signalling (STATs, PI‐3K, and PLCs) during the process of erythroid maturation. Human pro‐erythroblasts progressed in 4 days of culture with EPO into basophilic‐ (CD36high/CD235amedium, 24 h), polychromatic‐(CD36high/CD235ahigh, 48 h), and, finally, orthochromatic‐(CD36low/CD235ahigh, 72–96 h) erythroblasts. During this maturation, STAT‐1 was expressed up to the orthochromatic stage, expression of STAT‐5, as well as of its target proteins BclxL and IRF1, remained constant up to 48 h (polychromatic‐erythroblasts) but decreased by 96 h (orthochromatic‐erythroblasts), while that of STAT‐3 decreased constantly from 24 h on and became undetectable by 96 h. Expression of PI‐3K rapidly decreased with differentiation since only 50% of original protein levels were detected by 48 h. On the other hand, among the members of PLC families investigated, PLC β4 was not expressed, PLC β2, δ1, and γ2 were expressed at constant levels throughout the maturation process, while expression of PLC β3 and of PLC γ1 decreased, as PI‐3K, by 24 h and that of PLC β1 was induced by 6 h and became undetectable by 24 h. In conclusion, these data depict the dynamic signalling scenario associated with the maturation of erythroid cells and provide the first indication that members of PLC families (PLC β1, β3, and γ1) might be involved in the control of erythroid differentiation in humans.

Amplification of T cells from human cord blood in serum-deprived culture stimulated with stem cell factor, interleukin-7 and interleukin-2

Summary:We report the effects exerted by cytokine combinations, including stem cell factor (SCF), interleukin-7, interleukin-4 and interleukin-2, on the amplification of T cells from cord blood (CB) mononuclear cells cultured for 10–11 days under serum-deprived conditions. Of all the combinations investigated, SCF+interleukin-7 sustained the best fold increase (FI) of total nucleated cells (FI=6.4±1.17), amplifying preferentially CD4+ over CD8+ T-cell subsets (FI=4.72±0.79 vs 2.73±1.2, respectively, P<0.05). The addition of interleukin-2 to this combination did not significantly increase the total number of cells generated (FI=7.4±2.27), but allowed preferential amplification of CD8+ over CD4+ T cells (FI=6.04±0.14 vs 1.67±0.6, respectively, P<0.05). Single-strand conformation polymorphism analysis of the T-cell receptor Vβ-chain rearrangements expressed by the expanded T cells indicated that the complexity of the T-cell repertoire had increased after 10 days of culture in the presence of SCF and IL-7. Interestingly, a modest expansion (FI=8.67±1.5) of myeloid progenitor cells was also observed in these cultures. These results indicate that it is possible to expand specific T-cell subsets for adoptive immunotherapy without losing myeloid progenitor cells necessary for neutrophil recovery after CB transplantation, by modulating the cytokines added to the cultures.

Thymus-Independent T Cell Differentiation in Vitro

The generatation of large quantities of novel human T‐cell clones ex vivo would make a wide range of gene‐ and immuno‐therapies for tumours and viral infections possible. Several techniques have been described to generate, in vitro and in vivo (using xenogenic hosts), mature T cells from fetal‐neonatal and adult human CD34+ cells. All these techniques are cumbersome and cannot be easily translated into clinical protocols because they involve co‐cultivation of CD34+ cells with thymic fragments from either human or murine fetuses. We report that the mononuclear cells of human cord blood contain a cell population that supports the differentiation of CD34+ cells into CD4+ or CD8+ naive T cells in serum‐deprived cultures stimulated with stem cell factor and interleukin 7. CD4+ or CD8+ CD45RA+ TCRαβ+ T cells were continuously produced in vitro over a period of 20 d under these conditions. The generation of T cells in these cultures was a dynamic process and clones of T cells expressing new T‐cell receptor β‐chain rearrangments were generated over time. These results pave the way for the development of very simple culture conditions for ex‐vivo production of naive helper or cytotoxic T cells which could be very useful for gene‐ and immuno‐therapy of human diseases.

Protein kinase Cα is differentially activated during neonatal and adult erythropoiesis and favors expression of a reporter gene under the control of the Aγ globin-promoter in cellular models of hemoglobin switching

PKCα was found to be expressed (mRNA and protein) throughout the in vitro maturation of primary human erythroblasts but its activity (phosphorylation levels and nuclear localization) was consistently higher in cells derived from human neonatal rather than adult blood. Since the γ/γ + β globin expression ratio represented the major difference between neonatal and adult erythroblasts (58 ± 12 vs. 7 ± 3, respectively), we tested the hypothesis that PKCα might affect γ‐globin expression by measuring the levels of Aγ‐ or β‐promoter‐driven reporter activity in erythroid cells stably (GM979) or transiently (K562, primary adult and neonatal erythroblasts) transfected with a dual µLCRβprRlucAγprFluc reporter in the presence of transient expression of either the constitutively active (sPKCα) or catalytically inactive (iPKCα) PKCα. As further control, GM979 cells were incubated with the PKC inhibitor rottlerin (30 µM). In all the cells analyzed, sPKCα significantly increased (by two‐ to sixfold) the levels of luciferase activity driven by the Aγ‐promoter and the Aγ‐F/(Aγ‐F + 2β‐R) expression ratio. In GM979 cells, rottlerin inhibited (by 50%) the Aγ‐driven luciferase activity and the Aγ‐F/(Aγ‐F + 2β‐R) expression ratio. These results suggest that different PKC isoforms may exert ontogenetic‐specific functions in erythropoiesis and that modulation of PKCα might affect the activity of Aγ‐promoter‐driven reporters. J. Cell. Biochem. 101: 411–424, 2007.

Characterization of the T cell receptor repertoire of neonatal T cells by RT-PCR and single strand conformation polymorphism analysis

We have analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) the individual non-germ line configurations of the T cell receptor (TCR) Vβ chains expressed by T cells from eight individual cord blood specimens. cDNA from each cord blood was amplified using a common primer coupled with a primer specific for each of 22 variable elements of the Vβ chain family and the amplified fragments were separated under high resolution conditions. With cDNA from adult blood (as a control), all of the TCR chains were amplified as a smear consistent with the extensive polyclonality of adult T cells. In contrast, a heterogeneous pattern of amplification was observed with cDNAs from cord blood: only 26.7 ± 21.9% of the 22 Vβ chains analyzed were amplified as a smear. The majority of them were amplified as a discrete number of bands (up to 10) (in 68.2 ± 18.7% of samples) and some of them as a single fragment (4.0 ± 7.8%). Only one of the eight samples analyzed expressed the majority (72.7%) of its Vβ chains as a smear, consistent with an adult-like TCR repertoire. In conclusion, cord blood expressed, on average, a less complex TCR repertoire than adult blood. Bone Marrow Transplantation (2000) 26, 83–89.

Virus-free survival and down-regulation of CD4 in C8166 cells infected with human immunodeficiency virus type 1 at low density

Compared with other T cell lines, C8166 lymphocytes are particularly susceptible to human immunodeficiency virus (HIV) infection and the outcome is invariably cell death. The results reported in this study demonstrate that the virus-induced cytolysis is strongly dependent on the initial cell density of C8166 cultures. Cultures diluted to 50 to 500 cells/ml almost completely maintained their cell duplication rate and released infectious virus into the medium. HIV infection of diluted C8166 cells is a simple and easily reproducible procedure for obtaining persistently infected cultures. These cultures contained genomic and extragenomic HIV DNA, the latter being assayed by PCR for two-long terminal repeat circular forms. The status of persistent infection disappeared within 2 months. The recovery is due to the replacement of CD4 down-regulated infected cells by overgrowing uninfected cell variants, which are transcriptionally inactive for CD4. The mechanisms underlying the emergence of these variants in persistently infected cultures are considered.

TRANSPLANTATION AND CELLULAR ENGINEERING: Compensated variability in the expression of globin‐related genes in erythroblasts generated ex vivo from different donors

BACKGROUND: Ex vivo generated erythroblasts are being evaluated for transfusion. Expression of balanced levels of globin mRNA is essential for normal red blood cell function and survival but it is unknown whether the expression of the globin genes in ex vivo expanded cells is balanced.

Compensated variability in the expression of globin-related genes in erythroblasts generated ex vivo from different donors

BACKGROUND: Ex vivo generated erythroblasts are being evaluated for transfusion. Expression of balanced levels of globin mRNA is essential for normal red blood cell function and survival but it is unknown whether the expression of the globin genes in ex vivo expanded cells is balanced.