IntroductionIndoleamine 2,3-dioxygenase (IDO) is a key enzyme in the tryptophan metabolism that catalyzes the initial rate-limiting step of tryptophan degradation along the kynurenine pathway. 1 Tryptophan starvation by IDO consumption inhibits T-cell activation, 1,2 while products of tryptophan catabolism, such as kynurenine derivatives and O 2 -free radicals, regulate T-cell proliferation and survival. 1,3 Thus, IDO has been shown to exert an immunosuppressive activity, and cell populations, including regulatory dendritic cells (DCs) and bone marrow (BM)-derived mesenchymal stem cells (MSCs), expressing IDO have the capacity to suppress T-cell responses to auto-and alloantigens. 4,5 A wide variety of human solid tumors express IDO. 6 More recently, we demonstrated that also acute myeloid leukemia (AML) cells, but not their normal counterparts (ie, CD34 ϩ hematopoietic stem/progenitor cells [HSCs]), express an active IDO protein, which converts tryptophan into kynurenine and inhibits allogeneic T-cell proliferation. 7 Naturally arising CD4 ϩ CD25 ϩ Foxp3 ϩ T regulatory (T reg ) cells are known to suppress most types of immune response, 8,9 including antitumor immunity. [10][11][12][13] IDO is expressed and is functionally active in placenta, which, in turn, is infiltrated by CD4 ϩ CD25 ϩ T reg cells. [14][15][16] Moreover, Candida albicans infection increases the number of T reg cells because of IDO induction in host antigenpresenting cells (APCs). 17 In human cancers, tumor-draining lymph nodes contain IDO-expressing DCs that enhance T reg cell function. 18 These data suggest the close relationship between IDO activity and the occurrence of T reg cells, 19 but the mechanism governing the generation of T reg cells by IDO-expressing tumors is presently unknown.In the present study, we investigated whether the expression of IDO by AML cells may play a direct role in the development of T reg cells. Materials and methods CellsAll human samples were obtained after informed consent was signed, according to institutional guidelines. Approval was obtained from Bologna Hospital Ethical Committee. Buffy coats were obtained from healthy adults during the preparation of transfusion products. BM and/or peripheral blood (PB) samples including at least 70% leukemic cells were harvested from 76 patients with AML at diagnosis. CD3 ϩ and CD4 ϩ cells were purified from the mononuclear cell (MNC) fraction by MiniMacs high-gradient magnetic separation column (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions (purity of CD3 ϩ and CD4 ϩ cell populations was always greater than 95%). MSCs were generated from BM cells as previously reported. 20 Murine A20 and CT26 cell lines were obtained from the American Type Culture Collection (ATCC; Rockville, MD) and maintained in RPMI 1640 (Whittaker Bioproducts, Walkersville, MD) or MEM (Whittaker Bioproducts) supplemented with 10% FCS (Sera Lab, Crawley Down, United The online version of this manuscript contains a data supplement.The publication costs of ...
We designed a phase 1-2 study to evaluate the safety and the efficacy of increasing doses of bendamustine (160 mg/m 2 , 180 mg/m 2 , and 200 mg/m 2 given on days ؊7 and ؊6) coupled with fixed doses of etoposide, cytarabine, and melphalan (BeEAM regimen) as the conditioning regimen to autologous stem cell transplantation for resistant/relapsed lymphoma patients. Forty-three patients (median age, 47 years) with non-Hodgkin (n ؍ 28) or Hodgkin (n ؍ 15) lymphoma were consecutively treated. Nine patients entered the phase 1 study; no patients experienced a dose-limiting toxicity. Thirty-four additional patients were then treated in the phase 2. A median number of 6 ؋ 10 6 CD34 ؉ cells/kg (range, 2.4-15.5) were reinfused. All patients engrafted, with a median time to absolute neutrophil count > 0.5 ؋ 10 9 /L of 10 days. The 100-day transplantation-related mortality was 0%.
Viral-based techniques are the most efficient systems to deliver DNA into stem cells because they show high gene transduction and transgene expression in many cellular models. However, the use of viral vectors has several disadvantages mainly involving safety risks. Conversely, nonviral methods are rather inefficient for most primary cells. The Nucleofector technology, a new nonviral electroporation-based gene transfer technique, has proved to be an efficient tool for transfecting hard-to-transfect cell lines and primary cells. However, little is known about the capacity of this technique to transfect adult stem cells. In this study, we applied the Nucleofector technology to engineer human bone marrow-derived mesenchymal stem cells (hMSCs). Using a green fluorescent protein reporter vector, we demonstrated a high transgene expression level using U-23 and C-17 pulsing programs: 73.7% ؎ 2.9% and 42.5% ؎ 3.4%, respectively. Cell recoveries and viabilities were 38.7% ؎ 2.9%, 44.5% ؎ 3.9% and 91.4% ؎ 1.3%, 94.31% ؎ 0.9% for U-23 and C-17, respectively. Overall, the transfection efficiencies were 27.4% ؎ 2.9% (U-23) and 16.6% ؎ 1.4% (C-17) compared with 3.6% ؎ 2.4% and 5.4% ؎ 3.4% of other nonviral transfection systems, such as FUGENE6 and DOTAP, respectively (p < .005 for all comparisons). Nucleofection did not affect the immunophenotype of hMSCs, their normal differentiation potential, or ability to inhibit T-cell alloreactivity. Moreover, the interleukin-12 gene could be successfully transfected into hMSCs, and the immunomodulatory cytokine was produced in great amount for at least 3 weeks without impairment of its biological activity. In conclusion, nucleofection is an efficient nonviral transfection technique for hMSCs, which then may be used as cellular vehicles for the delivery of biological agents. STEM CELLS 2006;24:454 -461
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.