Thirteen patients with acute myeloid leukemia, 5 with active disease, 2 in molecular relapse, and 6 in morphologic complete remission (CR; median age, 62 years; range, 53-73 years) received highly purified CD56⁺CD3⁻ natural killer (NK) cells from haploidentical killer immunoglobulin-like receptor–ligand mismatched donors after fludarabine/cyclophosphamide immunosuppressive chemotherapy, followed by IL-2. The median number of infused NK cells was 2.74 × 10⁶/Kg. T cells were < 10⁵/Kg. No NK cell–related toxicity, including GVHD, was observed. One of the 5 patients with active disease achieved transient CR, whereas 4 of 5 patients had no clinical benefit. Both patients in molecular relapse achieved CR that lasted for 9 and 4 months, respectively. Three of 6 patients in CR are disease free after 34, 32, and 18 months. After infusion, donor NK cells were found in the peripheral blood of all evaluable patients (peak value on day 10). They were also detected in BM in some cases. Donor-versus-recipient alloreactive NK cells were shown in vivo by the detection of donor-derived NK clones that killed recipient's targets. Adoptively transferred NK cells were alloreactive against recipient's cells, including leukemia. In conclusion, infusion of purified NK cells is feasible in elderly patients with high-risk acute myeloid leukemia. This trial was registered at www.clinicaltrial.gov as NCT00799799.

Indoleamine 2,3-dioxygenase (IDO) is a novel immunosuppressive agent expressed in some subsets of normal and neoplastic cells, including acute myeloid leukemia (AML) cells. Here, we show that IDO expression correlates with increased circulating CD4⁺CD25⁺FOXP3⁺ T cells in patients with AML at diagnosis. In vitro, IDO⁺ AML cells increase the number of CD4⁺ CD25⁺ T cells expressing surface CTLA-4 and FOXP3 mRNA, and this effect is completely abrogated by the IDO inhibitor, 1-methyl tryptophan (1-MT). Purified CD4⁺CD25⁺ T cells obtained from coculture with IDO⁺ AML cells act as T regulatory (T_reg) cells because they do not proliferate, do not produce interleukin (IL)–2, and inhibit naive T-cell proliferation. Coculture with IDO⁺AML cells results in the conversion of CD4⁺CD25⁻ into CD4⁺CD25⁺ T cells, which is completely abrogated by 1-MT. Moreover, in mice, intrasplenic injection of IDO⁺ leukemia/lymphoma A20 cells induces the expansion of bona fide T_reg cells by conversion of CD4⁺CD25⁻ T cells; this effect is counteracted by 1-MT treatment. These data indicate that AML cells induce T-cell tolerance by directly converting CD4⁺CD25⁻ T cells into CD4⁺CD25⁺ T_reg cells through an IDO-dependent mechanism.

We designed a phase 1-2 study to evaluate the safety and the efficacy of increasing doses of bendamustine (160 mg/m², 180 mg/m², and 200 mg/m² 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 × 10⁶ CD34⁺ cells/kg (range, 2.4-15.5) were reinfused. All patients engrafted, with a median time to absolute neutrophil count > 0.5 × 10⁹/L of 10 days. The 100-day transplantation-related mortality was 0%. After a median follow-up of 18 months, 35 of 43 patients (81%) are in complete remission, whereas 6 of 43 relapsed and 2 of 43 did not respond. Disease type (non-Hodgkin lymphomas vs Hodgkin disease) and disease status at transplantation (chemosensitive vs chemoresistant) significantly influenced DFS (P = .01; P = .007). Remarkably, 4 of 43 (9%) patients achieved the first complete remission after receiving the high-dose therapy with autologous stem cell transplantation. In conclusion, the new BeEAM regimen is safe and effective for heavily pretreated lymphoma patients. The study was registered at European Medicines Agency (EudraCT number 2008-002736-15).

The inv(16) cytogenetic subtype of acute myeloid leukemia (AML) has a relatively good prognosis. Many patients achieve complete remission (CR). The prognostic uncertainty of negative qualitative reverse transcription–polymerase chain reaction (RT-PCR) assays suggests the need to identify prognostically significant critical thresholds by real-time RT-PCR. A reliable and sensitive (10⁻⁵) real-time RT-PCR assay was set up for the evaluation of relevant CBFβ-MYH11/ABL transcript ratios and was applied to the 21 patients with inv(16) AML routinely referred for cytogenetic and molecular monitoring in Seràgnoli Institute (Bologna, Italy) since 1990. Among the 18 patients who underwent ablative chemotherapy, all achieved CR with a 3-year disease-free survival probability of 63% (95% CI, 40%-87%) and no recorded events after 26 months. Five patients had relapses; 2 died of disease and 3 entered second CR. Analysis of the 125 bone marrow (or peripheral blood) samples studied by real-time RT-PCR showed that transcript ratios of samples taken during CR at any time before a relapse were always greater than 0.12%, whereas those of samples taken during first or second CR from patients who did not subsequently have relapses were always less than 0.25%. This suggests that transcript ratios greater than 0.25% may correspond to high risk for relapse, whereas ratios below 0.12% might indicate the patient is in a curable state. If confirmed, such thresholds could open the way to a new phase in post-CR therapeutic decision making for patients with inv(16) AML.

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 hM-SCs, 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.

Indoleamine 2,3-dioxygenase (IDO) is a key enzyme in the tryptophan metabolism, which 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 whereas products of tryptophan catabolism, such as kynurenine derivatives and O2 free radicals, regulate T-cell proliferation and survival.1, 3 Thus, IDO has a profound immunosuppressive activity. Accordingly, cell populations expressing IDO, including regulatory dendritic cells (DCs) and bone marrow (BM)-derived mesenchymal stem cells (MSCs), have the capacity to suppress T-cell responses to auto- and allo-antigens.4, 5 In these cells, the expression of IDO gene and protein is critically regulated by interferon (IFN)-.4, 5 Recent evidence has demonstrated that a wide variety of human solid tumors express the functionally active IDO protein, which acts as a mechanism of escape from the immune response.6 In particular, transfecting IDO into tumor cells prevents their rejection by pre-immunized hosts and the antitumor effect of IDO blockade is completely dependent on the presence of a fully competent immune system, thus suggesting that IDO acts by deregulating the host immune response. Despite the extensive work on solid tumor cells, no data are currently available for IDO expression and T-cell inhibitory function in human leukemic cells and normal hematopoietic stem cells (HSCs). To test whether BM-derived hematopoietic cells express IDO, we firstly analyzed total BM-derived mononuclear cells (MNCs) obtained from healthy volunteers. As shown in Figure 1a, BM-derived MNCs were negative for INDO expression, whereas INDO was detected in CD14+ monocytes purified from the peripheral blood (PB). Then, we analyzed highly purified CD34+ HSCs from BM of healthy donors. Figure 1b shows that 5/5 CD34+ cell samples did not express INDO. We then asked whether CD34+ cells could express IDO gene and protein upon exposure to optimal concentration of IFN-. CD34+ HSCs from healthy donors expressed INDO after incubation with IFN- (Figure 1c), but were negative for IDO protein, as evaluated by both immunocytochemistry (data not shown) and Western blot analyses (Figure 1d). Conversely, BM non-hematopoietic IFN--stimulated MSCs expressed IDO mRNA (Figure 1c) and protein (Figure 1d). These data suggest that BM-derived hematopoietic and non-hematopoietic cells follow different pathways for IDO expression, perhaps owing to a different susceptibility to IFN- modulation. In addition, normal IFN--stimulated CD34+ cells express IDO mRNA, but not IDO protein, thus indicating a post-transcriptional regulation of IDO protein expression in normal HSCs. To test whether leukemic cells express IDO, primary AML cells collected from 76 AML patients at diagnosis (Supplementary Table 1) were tested for IDO expression. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated that 40/76 (52%) AML samples were positive for INDO mRNA. Accordingly, IDO protein was expressed only in RT-PCR-positive AML samples as evaluated both by immunocytochemistry (data not shown) and Western blot analyses (Figure 1e). Similar to normal CD34+ cells, IDO- AML cells expressed INDO mRNA, but not IDO protein when exposed to optimal concentrations of IFN- (Figure 1f and g). IFN- stimulation of IDO+ AML cells did not upregulate INDO mRNA (Figure 1f). Interestingly, the expression of IFN- receptor (CD119) was significantly downregulated in IDO+ as compared to IDO- AML cells (Figure 1h). The median fluorescence intensity (MFI) of IDO- and IDO+ AML cells was 8710 and 3214, respectively (P=0.01). Taken together, these data demonstrate that, similar to solid tumors,6 a significant portion of AML samples shows a constitutive expression of IDO. Similarly to normal HSCs, a subset of AML samples lacks IDO expression, which can be induced after exposure to IFN- only at molecular, but not at protein level. Thus, the differential expression of IDO among normal CD34+ HSCs and leukemic cells supports the hypothesis that, in a subset of AML patients, the constitutive expression of IDO protein is associated with the leukemic transformation and may play a role in leukemia development. A further set of experiments was performed to test the functional activity of IDO protein expressed by AML cells. IDO+ and IDO- AML cells were cultured for 48 h and then supernatants were analyzed for L-tryptophan and L-kynurenine concentrations. In these experiments, IFN--stimulated MSCs were used as positive control. As shown in Figure 2a and b, IDO from AML cells was functionally active, resulting in the reduction of tryptophan and the increase of L-kynurenine in the culture medium. Accordingly, IDO- AML cells showed a very little capacity of tryptophan depletion from culture medium and, more importantly, they were not capable to produce kynurenine metabolite. To test whether IDO+ and IDO- AML cells were different in their ability to stimulate T-cell proliferation, AML cells were used as stimulators for allogeneic CD3+ T cells in a standard mixed lymphocyte reaction (MLR). As shown in Figure 2c, T-cell proliferation induced by IDO- AML cells was slightly, albeit not significantly, increased over that observed with IDO+ AML cells. However, in agreement with our data on IDO expression, the addition of increasing concentrations of 1-D-MT resulted in a significant increase of T-cell proliferation only when IDO+, but not IDO- AML cells were used as stimulators (P=0.02). Similar results were observed when the L isomer of 1-MT, which is considered a better IDO-inhibitor than 1-D-MT, was used in MLR cultures (Figure 2d). Moreover, tryptophan depletion from day 5 MLR supernatants was significantly increased in presence of IDO+ AML cells as compared to IDO- AML cells (Figure 2e). Frequencies of INDO gene expression among French-American-British (FAB) subgroups were analyzed. There was a higher, but not statistically...