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Objective: Interleukin 15 (IL)-15 controls proliferation and survival of T cells, but its effects and the underlying cellular regulation are not well understood. Previous studies have focused on its effects on short-term T-cell cultures. In view of the potential problems associated with using IL-2 alone in adoptive immunotherapy protocols, we investigated the impact of IL-15 on T-cell cultures and the global transcriptional effects it elicits in such cultures., Materials and Methods: DNA microarrays and flow cytometry were used to examine the differential effect of 20 ng/mL IL-15 on primary serum-free T-cell cultures activated and cultured in the presence of IL-2. Quantitative reverse transcriptase polymerase chain reaction confirmed select microarray data., Results: IL-15 significantly increased ex vivo expansion of primary human T cells over the entire 11-day expansions without affecting viability. The 1133 genes were consistently differentially expressed among three donor samples. Ontological analysis demonstrated that IL-15 increases expression of genes involved in inflammatory response (e.g., tumor necrosis factor [TNF]-alpha, Oncostatin M, CD40L, and CD33) and apoptosis (e.g., TNF-related apoptosis-inducing ligand). IL-15 also induced expression of four suppressors of cytokine signaling (SOCS) family genes (SOCS1-3, cytokine-inducible SH2-containing protein), which are classical negative regulators of cytokine signaling. IL-15 strongly suppressed the expression of inhibitory natural killer cell receptor genes, including three C-type lectins (KLRB1, KLRC1, and KLRD1), as well as IL-7Ra and Granzyme H. Finally, IL-15 induced differential expression of TNF receptor superfamily members (CD27 and CD30)., Conclusion: These findings suggest that exogenous IL-15 may have a potential role in adoptive immunotherapy by both enhancing proliferation and modulating functionality during ex vivo T-cell expansion.

Objective: Mylotarg (gemtuzumab ozogamicin [GO]) has recently been introduced as a novel CD33-targeting drug in clinical hematology. However, despite efficacy, GO produces significant side effects including an infusion syndrome. We have recently shown that mast cells (MCs) and basophils (BAs) express CD33. In the present study, we investigated the effects of GO on growth and mediator secretion in MCs and BAs., Methods: Growth-inhibitory effects of GO on neoplastic MCs (HMC-1) and BAs (KU812) as well as cord blood-derived MC and BA progenitor cells were determined by counting cell numbers and the numbers of apoptotic cells. The amount of histamine secreted from primary MCs and BAs was measured by radioimmunoassay after incubation of cells with GO alone or GO together with an anti-immunoglobulin E (IgE) antibody., Results: MCs and BAs as well as HMC-1 cells and KU812 cells were found to express CD33 mRNA and the CD33 protein. GO was found to inhibit the growth of HMC-1 cells and KU812 cells as well as stem cell factor-dependent differentiation of MCs and interleukin-3-induced growth of BAs from their cord blood-derived progenitors. The GO-induced inhibition of growth of neoplastic cells was found to be associated with induction of apoptosis. GO neither induced secretion of histamine from MCs or BAs nor upregulated the anti-IgE-induced release of histamine in these cells., Conclusions: GO counteracts growth of normal and neoplastic MCs and BAs without inducing rapid release of histamine. The exact value of GO as a targeted drug for the treatment of high-grade MC or BA neoplasms remains to be determined.

Objective: To evaluate the effects of granulocyte colony-stimulating factor (G-CSF) on the in vitro sensitivity of acute myeloid leukemia (AML) cell lines and primary AML blast cells to gemtuzumab ozogamicin (GO)., Materials and Methods: AML cell lines and primary blasts from 10 patients with AML were first incubated for 72 hours in the presence of G-CSF (5 or 100 ng/mL) and then exposed to increasing concentrations of GO (1-1,000 ng/mL) for an additional 72 hours., Results: Pretreatment with G-CSF translated into significant enhancement of GO-induced cytotoxicity in the GO-sensitive HL-60 and NB-4 cells. Conversely, the response of GO-insensitive KG-1a, TF-1, and K562 cells was unaffected by in vitro priming with G-CSF. In vitro exposure to G-CSF augmented GO-induced apoptosis in 7 of 10 primary AML samples and rendered blast cells from three refractory patients sensitive to killing effect of GO. The G-CSF-induced increase of the cytocidal activity of GO was independent of effects on the cell cycle and on the expression levels of CD33 antigen. Of potential interest, G-CSF induced dose-dependent inhibition of P-glycoprotein (P-gp/ABCB1) function in the GO-sensitive HL-60 and NB-4 cells and in blasts from three patients with AML that we tested., Conclusion: Collectively, our findings point to G-CSF as a potential sensitizing agent that can be exploited therapeutically to improve the clinical efficacy of GO.

Objective: Neutrophil receptors for the Fc portion of IgG (FcgammaR) trigger immune responses following cross-linking by IgG-coated foreign particles or immune complexes. Membrane-associated CD45, a protein tyrosine phosphatase termed leukocyte common antigen, has been shown to be essential for antigen receptor kinase mediated signaling in lymphocytes, and we hypothesized that CD45 may play a similar role in FcgammaR-mediated signaling and immune function in human neutrophils., Methods: The experimental approach was that of cell surface molecule ligation via cross-linking with specific antibodies. Antibody dependent cellular cytotoxicity (ADCC) was assessed using a single-cell plaque assay and IL-6 production measured using ELISA. Tyrosine phosphorylation levels were assessed with anti-phospho-tyrosine blots and F-actin polymerization by flow cytometry and confocal microscopy., Results: Neutrophils pretreated with anti-CD45 had a reduced ability to perform ADCC compared to untreated neutrophils. FcgammaRIIa cross-linking resulted in significantly increased concentrations of secreted IL-6 compared to untreated neutrophils, and IL-6 production was further enhanced by cocross-linking CD45 with FcgammaRIIa. Cross-linking CD45 alone also induced IL-6 production. FcgammaRIIa cross-linking resulted in increased protein tyrosine phosphorylation and F-actin polymerization in neutrophils. Cocross-linking CD45 with FcgammaRIIa resulted in abrogation of FcgammaRIIa mediated tyrosine phosphorylation and F-actin polymerization., Conclusions: These data provide evidence that CD45 can regulate or enhance the stimulation and function of human neutrophils mediated through FcgammaR(s). In addition, CD45 ligation may play an essential role in cytokine induction pathways that lead to inflammatory reactions in vivo.

Recent studies show that apoptosis is important for the resolution of chronic inflammation. Using a human myeloblastic leukemia cell line, EoL-1, we investigated the effect of interferon-gamma (IFN-gamma), which differentiates EoL-1 into monocyte/macrophage-like cells on Fas antigen (Fas)- and tumor necrosis factor-alpha (TNF alpha)-induced apoptosis. Both TNF and anti-Fas monoclonal antibody (CH-11) induced apoptosis of EoL-1 cells. Pretreatment with IFN-gamma for 72 hours enhanced the CH-11-induced apoptosis with up-regulation of Fas. However, the treatment markedly inhibited the TNF-induced apoptosis. In flow cytometric analysis, EoL-1 expressed two types of tumor necrosis factor receptors (TNFR1 and TNFR2), and the expression of TNFR2 but not of TNFR1 was up-regulated significantly after the IFN-gamma treatment. The TNF-induced apoptosis was mimicked by a TNFR1 stimulating antibody (htr-9), and was reversed by a TNFR1 blocking antibody (H398). Although the TNFR1-mediated cytotoxic signal was not affected by IFN-gamma pretreatment, blocking TNFR2 by a specific antagonistic antibody (utr-1) canceled the inhibitory effect of IFN-gamma. In conclusion, TNF-induced apoptosis was mediated preferentially by TNFR1, and the anti-apoptotic effect of IFN-gamma was result from up-regulated TNFR2 in EoL-1 cell line. This cell line is a useful model to provide new insights into crosstalk among Fas/FasL-, TNF-, and IFN-gamma-mediated signaling.

Transforming growth factor beta (TGF-beta) is a highly conserved peptide with growth-inhibitory activity in multiple normal and transformed cell types. Signal transduction is mediated through the receptor complex, consisting of two active seronine or threonine kinases (TGF-beta-receptor I and II) and the receptor-associated proteins betaglycan (TGF-beta-receptor III) and endoglin. In this study, we assessed the analysis of the role of TGF-beta and the transcription of the genes for TGF-beta and its receptor in highly purified leukemic B-cell precursors (BCPs) of patients with common acute lymphoblastic leukemia (cALL). Leukemic BCPs were positive for gene transcription of TGF-beta (9/9), the TGF-beta-receptor I (9/9), the TGF-beta-receptor II (6/6), betaglycan (5/6), and endoglin (6/6). Incubation with TGF-beta significantly reduced the cell viability of leukemic BCPs by a mean of 45% (p = 0.0009). The reduction of cell viability was associated with the induction of apoptosis by a mean of 31%. TGF-beta caused significant suppression of the S phase (p = 0.002) and accumulation in the G0/G1 phase (p = 0.0005). It also reduced expression of the adhesion surface receptor CD18 and the Fas antigen CD95 from 58% to 40% and from 48% to 27%, respectively. The data indicate that TGF-beta is a negative growth signal in leukemic BCPs and point to an additional role of TGF-beta as an immunomodulatory cytokine, suggesting a complex role of TGF-beta in the leukemogenesis of cALL.