Immunomagnetic isolation of CD4+CD25+FoxP3+ natural T regulatory lymphocytes for clinical applications.

Although CD4(+)/CD25(+) T regulatory cells (T(regs)) are a potentially powerful tool in bone marrow transplantation, a prerequisite for clinical use is a cell-separation strategy complying with good manufacturing practice guidelines. We isolated T(regs) from standard leukapheresis products using double-negative selection (anti-CD8 and anti-CD19 monoclonal antibodies) followed by positive selection (anti-CD25 monoclonal antibody). The final cell fraction (CD4(+)/CD25(+)) showed a mean purity of 93.6% +/- 1.1. Recovery efficiency was 81.52% +/- 7.4. The CD4(+)/CD25(+bright) cells were 28.4% +/- 6.8. The CD4(+)/CD25(+) fraction contained a mean of 51.9% +/- 15.1 FoxP3 cells and a mean of 18.9% +/- 11.5 CD127 cells. Increased FoxP3 and depleted CD127 mRNAs in CD4(+)CD25(+)FoxP3(+) cells were in line with flow cytometric results. In Vbeta spectratyping the complexity scores of CD4(+)/CD25(+) cells and CD4(+)/CD25(-) cells were not significantly different, indicating that T(regs) had a broad T cell receptor repertoire. The inhibition assay showed that CD4(+)/CD25(+) cells inhibited CD4(+)/CD25(-) cells in a dose-dependent manner (mean inhibition percentages: 72.4 +/- 8.9 [ratio of T responder (T(resp)) to T(regs), 1:2]; 60.8% +/- 20.5 (ratio of T(resp) to T(regs), 1:1); 25.6 +/- 19.6 (ratio of T(resp) to T(regs), 1:0.1)). Our study shows that negative/positive T(reg) selection, performed using the CliniMACS device and reagents, enriches significantly CD4(+)CD25(+)FoxP3(+) cells endowed with immunosuppressive capacities. The CD4(+)CD25(+)FoxP3(+) population is a source of natural T(reg) cells that are depleted of CD8(+) and CD4(+)/CD25(-) reacting clones which are potentially responsible for triggering graft-versus-host disease (GvHD). Cells isolated by means of this approach might be used in allogeneic haematopoietic cell transplantation to facilitate engraftment and reduce the incidence and severity of GvHD without abrogating the potential graft-versus-tumour effect.

Haematopoietic and stromal stem cell regulation by extracellular matrix components and growth factors.

During human embryogenesis, differentiation of haematopoietic stem cells (HSCs) and their progeny is regulated spatially and temporally. In the adult, hemopoiesis is restricted to bone marrow (BM) which contains HSCs residing within the so-called 'niches'. These are microenvironments consisting of extracellular matrix (ECM) macromolecules (mainly glycosaminoglycans, proteoglycans, fibronectin and collagens) and stromal cells that act in concert to keep HSCs in quiescence or to promote their growth and differentiation, since BM stromal cells secrete specific growth factors acting on responsive stem cells. Haematopoietic precursors also secrete numerous regulatory molecules as fibroblast growth factors (FGF), interleukin 1 (IL1), and transforming growth factor-beta1 (TGFbeta1), regulating in an autocrine and/or paracrine manner the various stages of normal hematopoiesis. Although the majority of stem cells are quiescent and do nor respond to external signals, a few active stem cells responde to paracrine produced growth factors and differentiate into the more committed CD34+ haematopoietic stem cell or into a mesenchymal stem cell, which generate even more specified tissue. This review focuses on the role of both ECM molecules and growth factors that form a dynamic, interactive system crucial for lineage commitment and amplification. In this perspective, we recently described the pivotal role of ECM, FGF and TGFbeta on the GM-490 phenotype, which is a cell line established from the bone marrow of a patient with acute lymphoblastic leukemia. Our findings indicated the GM-490 cell line possess characteristics of both haematopoietic and mesenchymal precursors.

Topological organization of NADPH-oxidase in haematopoietic stem cell membrane: preliminary study by fluorescence near-field optical microscopy.

The aim of this study was to characterize the local distribution and organization of the plasma membrane NADPH-oxidase (NOX) in human haematopoietic stem cell (HSC) by means of the fluorescence scanning near-field optical microscopy approach. The presence of NOX in haematopoietic stem cells is thought to have a functional role as O(2) sensor and/or as low-level reactive oxygen species (ROS) producer to be used as redox messenger for controlling cell growth and differentiation. Given the harmful potential of ROS, a fine-tuning of NOX activity is needed. The high resolution imaging of haematopoietic stem cell membrane obtained in this study combined with the immunodetection of NOX indicates for this the occurrence of a cluster-organized structure. These membrane 'rafts'-like micro-compartments may constitute localized protein aggregates whereby the assembly/activation of the NOX components are functionally integrated with upstream factors constituting signal-transduction platforms.

Recurrent primary plasmacytoma of the eyelid with rapid regional metastasis.

In this case, originally reported as primary eyelid plasmacytoma, the tumor recurred on the same eyelid within 2 years of surgery. No plasma cell infiltration was observed at bone marrow biopsy. No serum or urinary monoclonal component was detected at immunofixation. Histology and immunohistochemistry confirmed plasma cell infiltration. Tumor cell clonality was determined by immunohistological staining; cells were positive for kappa light chain like the first eyelid tumor. Surgery was followed by radiotherapy. Twenty months later, biopsy of one enlarged right cervical lymph node showed massive diffuse infiltration of atypical plasma cells (CD20(-), CD79a(+), CD138(+), MUM1/IRF4(+)). Given the rapid diffusion to lymph nodes and the appearance of the monoclonal component, the lymph node was removed surgically. No adjuvant chemotherapy was given. Unexpectedly, the serum monoclonal component normalized. No plasma cell infiltration was observed at bone marrow biopsy. As this case might be a particularly slow-progressing extra-medullary plasmacytoma, this study recommends closely monitored follow-ups so that the aggressive form can be treated in time.

Effect of a p210 multipeptide vaccine associated with imatinib or interferon in patients with chronic myeloid leukaemia and persistent residual disease: a multicentre observational trial.

BACKGROUND: Although imatinib is the standard treatment for chronic myeloid leukaemia, not all patients reach complete cytogenetic remission (CCR) and most maintain detectable disease at the molecular level. We investigated whether a vaccine targeting the BCR-ABL-derived p210 fusion protein was an active and specific immunotherapy. METHODS: We recruited 16 patients who had chronic myeloid leukaemia (with the b3a2 fusion point of p210), stable residual disease, a minimum treatment of 12 months of imatinib or 24 months of interferon alfa, and no further reduction of residual disease for at least 6 months preceding enrollment. They were given six vaccinations with a peptide vaccine derived from the sequence p210-b3a2 plus molgramostim and QS-21 as adjuvants (CMLVAX100) before assessment of immunological and disease response, which included detecting amounts of b3a2 transcripts by standardised quantitative real-time reverse-transcriptase PCR. RESULTS: Of ten patients on imatinib, nine started CMLVAX100 having had a median of 10 months' stable cytogenetic disease (median 10% Philadelphia-chromosome-positive metaphases), whereas one started in stable CCR. All patients' cytogenetic responses improved after six vaccinations, with five reaching CCR. Notably, three of these five patients also had undetectable amounts of b3a2 transcript (BCR-ABL:beta2 microglobulin ratio <0.00001). Six patients on interferon alfa treatment with a median of 17 months' stable residual disease (median 13% Philadelphia-chromosome-positive cells) were also vaccinated. All but one had improved cytogenetic responses, and two reached CCR. Overall, we recorded peptide-specific delayed-type hypersensitivity (in 11 of 16 patients), CD4 cell proliferation (13 of 14 assessed), and interferon gamma production (five of five assessed). INTERPRETATION: Addition of CMLVAX100 to conventional treatment in patients with chronic myeloid leukaemia might favour further reduction of residual disease and increase the number of patients reaching a molecular response.

Arsenic trioxide (ATO) and MEK1 inhibition synergize to induce apoptosis in acute promyelocytic leukemia cells.

Recent studies suggest that components of the prosurvival signal transduction pathways involving the Ras-mitogen-activated protein kinase (MAPK) can confer an aggressive, apoptosis-resistant phenotype to leukemia cells. In this study, we report that acute promyelocytic leukemia (APL) cells exploit the Ras-MAPK activation pathway to phosphorylate at Ser112 and to inactivate the proapoptotic protein Bad, delaying arsenic trioxide (ATO)-induced apoptosis. Both in APL cell line NB4 and in APL primary blasts, the inhibition of extracellular signal-regulated kinases 1/2 (ERK1/2) and Bad phosphorylation by MEK1 inhibitors enhanced apoptosis in ATO-treated cells. We isolated an arsenic-resistant NB4 subline (NB4-As(R)), which showed stronger ERK1/2 activity (2.7-fold increase) and Bad phosphorylation (2.4-fold increase) compared to parental NB4 cells in response to ATO treatment. Upon ATO exposure, both NB4 and NB4-As(R) cell lines doubled protein levels of the death antagonist Bcl-xL, but the amount of free Bcl-xL that did not heterodimerize with Bad was 1.8-fold greater in NB4-As(R) than in the parental line. MEK1 inhibitors dephosphorylated Bad and inhibited the ATO-induced increase of Bcl-xL, overcoming ATO resistance in NB4-As(R). These results may provide a rationale to develop combined or sequential MEK1 inhibitors plus ATO therapy in this clinical setting.

Mesenchymal cells: a vehicle for gene therapy.

T cell-depleted allogeneic stem cell transplantation is associated with delayed immunological reconstitution. Bone marrow stroma and interleukin 7 (IL-7) regulate homeostasis of T lymphocytes. We engineered human stromal cells with a retroviral vector containing the IL-7 gene and studied in vitro effects on T cells. Human stromal cells were successfully transduced and generated a layer that was morphologically and phenotypically normal. IL-7-engineered stromal cells conserve the biological properties of unmanipulated stromal cells. Through their production of IL-7, they enhance survival and homeostatic proliferation of naive T cells. Because of this cytokine production, they might be an ideal vehicle for gene therapy aimed at supporting lymphopoiesis in the T cell-deficient host.

Downmodulation of ERK activity inhibits the proliferation and induces the apoptosis of primary acute myelogenous leukemia blasts.

MAP kinase/ERK kinase (MEK)-extracellular signal-regulated kinase (ERK) kinases are frequently activated in acute myelogenous leukemia (AML), and can have prosurvival function. The purpose of this study was to induce downmodulation of MEK-ERK activation in AML primary blasts in order to detect the effect on cell cycle progression and on the apoptosis of leukemic cells. We investigated 14 cases of AML with high ERK 1/2 activity and four cases with undetectable or very low activity. After 24 h incubation of the AML blasts with high ERK activity using PD98059 (New England BioLabs, Beverly, MA, USA), a selective inhibitor of MEK1 phosphorylation, at concentrations of 20 and 40 microM, we observed a strong decrease in the levels of ERK1/2 activity. A significant decrease of blast cell proliferation compared with untreated controls was found. In contrast, the proliferation of blast cells that expressed low or undetectable levels of ERK activity was not inhibited. Time-course analysis demonstrated that the downmodulation of MEK1/2, ERK1 and ERK2 dual-phosphorylation was evident even after 3 h of treatment with 20 and 40 microM. The cleavage of poly(ADP-ribose) polymerase (PARP), an early sign of apoptosis, appeared after 18 h of PD98059 treatment at concentrations of 20 and 40 microM in eight of the 14 cases. After 24 h of treatment, cleaved PARP appeared in all 14 cases. Time-course analysis of cell cycle progression and apoptosis showed that PD98059 induced a G1-phase accumulation with low or undetectable levels of apoptosis after 24 h incubation; after 48 and 72 h incubation, a significant increase of apoptosis was observed. Thus, the primary effect of ERK downmodulation was a cell cycle arrest followed by the apoptosis of a significant percentage of the leukemic blasts. The preclinical model of leukemia treatment reported in this paper makes further comment with regard to MEK1 inhibition as a useful antileukemic target, and encourages the conducting of in vivo studies and clinical investigations.

Haploidentical stem cell transplantation for acute leukemia.

PREMISE: Since March 1993, 133 patients with high-risk acute leukemia (66 AML, 67 ALL) have received a megadose of T-cell depleted hematopoietic stem cells. The 1993-95 conditioning protocol included TBI, thiotepa, ATG and CY for 36 patients who received an inoculum made up of lectin-separated bone marrow and PBPCs. After 1995, to minimise the extra-hematological toxicity of the conditioning and eliminate GvHD, we substituted fludarabine for CY in the conditioning and PBPCs were depleted of T-cells by a positive selection of the CD34+ cells using CellPro (n=44 patients) or, since January 1999, CliniMacs (n = 53 patients). A later modification to the protocol in January 1999 was the suspension of post transplant G-CSF. WORK IN PROGRESS: We report here the results in the last 53 acute leukemia patients all of whom were transplanted under our modified protocol. Ages ranged from 9 to 62 years with a median of 38 years for the 33 patients with AML and 23 for the 20 with ALL. All were at high risk because 25 were actually in relapse at transplant, 16 were in second or later CR and even the 12 patients in CR1 were at high risk because of the unfavourable prognostic features. Overall 52/53 patients (98%) engrafted. The TBI-Fludarabine-based conditioning was well tolerated even in the 14 patients between 45 and 62 years of age. There was no veno-occlusive disease of the liver and the incidence of severe mucositis was low. Even though no post-transplant immunosuppressive therapy was given, acute GvHD grade > or = II occurred in only 4 cases and only one progressed to chronic GvHD. Overall, 16 patients (30%) have died of non-leukemic causes. Relapses occurred mainly in patients who were already in relapse at transplant (12/25). Only 3 of the 28 who were in any CR at transplant have so far relapsed. As our group has already shown, donor-vs-recipient NK cell alloreactivity exerts a specific graft-vs-AML effect in the absence of GvHD. In fact, leukemia relapse was largely controlled in AML recipients whose donor was NK alloreactive, with only 2 out of 16 relapsing. To date, 13 of 18 AML (72%) and 5 of 10 ALL (50%) who were in any CR at transplant, survive disease-free while 4 of the 15 patients (16%) in relapse at transplant survive. The probability of event-free survival for patients transplanted in CR is 60% in the 18 AML patients and 38% in the 10 ALL. The probability of EFS was significantly better in the 16 AML patients whose transplant included donor vs recipient NK cell alloreactivity than in those whose transplant did not (70% vs 7%). In conclusion, given our current results, the most suitable candidate for the full haplotype mismatched transplant should be in early stage disease and selection of an NK alloreactive donor is recommended.

Haploidentical stem cell transplantation in leukemia.

In acute leukemia patients, infusing a megadose of extensively T-cell-depleted hematopoietic stem cells after an immuno-myeloablative conditioning regimen ensures sustained engraftment of full-haplotype mismatched transplants without graft-vs-host disease. Besides the conditioning regimen and the megadose of stem cells donor natural killer cell alloreactivity also plays a role in facilitating engraftment and in preventing relapse. Since our first successful pilot study, our efforts have concentrated on developing new conditioning regimens, optimizing the graft processing and improving the post-transplant immunological recovery. The results we have so far achieved in 112 very high-risk acute leukemia patients show that haploidentical transplantation is now a clinical reality. Because virtually all patients in need of a hematopoietic stem cell transplant have a full-haplotype mismatched donor, who is immediately available, a T-cell depleted mismatched transplant should be offered, not as a last resort, but as a viable option to high risk acute leukemia patients who do not have, or cannot find, a matched donor.

Prenatal diagnosis of genetic abnormalities using fetal CD34+ stem cells in maternal circulation and evidence they do not affect diagnosis in later pregnancies.

In the present study, we report a new method for enrichment and analysis of fetal CD34+ stem cells after culture in order to determine whether it is feasible for noninvasive prenatal diagnosis. We also determined whether fetal CD34+ stem cells persist in maternal blood after delivery and assessed whether they have an impact on noninvasive prenatal diagnosis of genetic abnormalities. Peripheral blood samples were obtained from 35 pregnant women, 13 non-pregnant women who had given birth to male offsprings, 12 women who had never been pregnant, and eight pregnant women with male fetuses. CD34+ stem cells were enriched and either cultured for prenatal diagnosis or analyzed with fluorescence in situ hybridization (FISH)/polymerase chain reaction (PCR) to determine peristance in maternal blood. Fetal/maternal cells can be isolated and grown "in vitro" to provide enough cells for a more accurate fetal sex or aneuploid prediction than is provided by unenriched and uncultured CD34+ stem cells. The presence of fetal cells in maternal blood samples from mothers who had given birth to male offspring was found in 3 of 13 blood samples. PCR was positive for Y chromosome in one woman who had never been pregnant. Analysis of cultured CD34+ stem cells from mothers with Y PCR positivity did not detect any male cells in any samples. Even if PCR positivity is due to persistence of fetal stem cells from previous pregnancies, it does not seem to affect this new system of enrichment, culture, and FISH analysis of CD34+ fetal stem cells.

Clinical activity and safety of combination immunotherapy with IFN-alpha 2a and Rituximab in patients with relapsed low grade non-Hodgkin's lymphoma.

BACKGROUND AND OBJECTIVES: To determine the clinical activity and safety of the combination immunotherapy of the chimeric anti-CD20 antibody, Rituximab, and Interferon (IFN)- alpha 2a DESIGN AND METHODS: Sixty-four patients with relapsed low-grade or follicular B-cell non Hodgkin's lymphoma received 4 infusion of Rituximab (375 mg/m(2) x dose) after priming and simultaneous treatment with IFN- alpha 2a. RESULTS: The overall response rate was 70% with 33% complete responses. Median for duration of response is 19 months, after a median follow-up of 22 months. By univariate analysis none of the most common prognostic factors predicted for response to therapy. After treatment 10 patients become bcl-2 negative in the bone marrow, but no correlation between molecular and clinical response was found. Fifty-three patients (83%) had drug related or unknown origin adverse events. The number of adverse events per patient varied from 1 to 21. Considering all 272 events, 231 (85%) were grade 1 or 2, 36 (13%) grade 3 and 5 (2%) grade 4. Twenty-three patients required reduction in the dose and/or short discontinuation of IFN treatment, either during priming or subsequent treatment. The most frequent adverse events were leukopenia, fever, neutropenia, hypotension and thrombocytopenia. INTERPRETATION AND CONCLUSIONS: this report shows that combination immunotherapy Rituximab + IFN- alpha 2a is active and relatively well tolerated. The overall response rate of 70% and the median duration remission of 19 months compare favorable with the results obtained with Rituximab alone in similar subset of patients. Randomized trials, investigating Rituximab versus combination immunotherapy are needed.

Results of a prospective study with high-dose etoposide, thiotepa and carboplatin and peripheral blood stem cell rescue for high-risk stage II-IIIA and selected stage IV breast cancer patients.

AIMS AND BACKGROUND: To investigate the safety and efficacy of a high-dose chemotherapy regimen with etoposide, carboplatin and thiotepa in high-risk stage II-IIIA breast cancer and in responsive metastatic patients. STUDY DESIGN: From April 1992 to December 1998, 24 patients with high-risk stage II-IIIA breast cancer (> or = 9 positive nodes) and 9 responsive metastatic patients were enrolled in the trial. After induction chemotherapy with an anthracycline-based regimen, peripheral blood stem cells were mobilized with cyclophosphamide (7 g/m2) and G-CSF (5-16 microg/kg/s.c./day). The high-dose chemotherapy regimen consisted of etoposide (1000 mg/m2), carboplatin (800 mg/m2) and thiotepa (500 mg/m2). At the end of the high-dose chemotherapy, all stage II-IIIA patients received radiotherapy to the breast or chest wall and draining nodes; stage IV patients were irradiated to sites of disease, if feasible. All ER+ and/or PgR+ patients were treated with hormone therapy. RESULTS: For stage II-IIIA high-risk patients, the median follow-up was 4.36 years (range, 1.93-6.94), and the Kaplan-Meier estimate at 5 years of disease-free survival and overall survival was 54.8 +/- 11% SE and 76.73 +/- 9.4% SE, respectively. For metastatic patients, the median follow-up was 4.93 years (range, 4.15-7.95), and the Kaplan-Meier estimate at 5 years of progression-free survival and overall survival was 22.2 +/- 13.9% SE and 76.2 +/- 14.8% SE, respectively. No treatment-related deaths were observed. CONCLUSIONS: Our results are comparable to those obtained in other high-dose chemotherapy trials but do not seem to be superior to conventional-dose therapy given to similar patients.

Allogeneic transplantation across the HLA barriers.

In high-risk acute leukemia patients, a 10-fold increase in the dose of extensively T-cell-depleted hematopoietic stem cells ensures sustained full-donor engraftment of one-haplotype-mismatched transplants without graft-vs.-host disease. Since our first successful pilot study, which exploited the principle of a megadose stem cell transplant, our efforts have concentrated on developing new conditioning regimens, optimizing graft processing and improving the post-transplant immunologic recovery. The results so far achieved in more than 100 high-risk acute leukemia patients show that haploidentical transplantation is now a clinical reality. Because virtually all patients in need of a hematopoietic stem cell transplant have a full-haplotype-mismatched family donor, a T-cell-depleted mismatched transplant can be offered with curative intent, thus extending allogeneic transplantation procedures to virtually all candidates.

Expression and activation of SHC/MAP kinase pathway in primary acute myeloid leukemia blasts.

INTRODUCTION: We report the results of a study investigating signaling proteins in 26 cases of primary acute myelogenous leukemia. We studied the Shc adaptor proteins p52/p46Shc, which can activate the RAS/Mitogen Activated Protein kinase pathway, p66Shc which is uncoupled from RAS/MAP kinases and the MAP kinase family members Extracellular signal Regulated Kinase (ERK) and c-Jun NH2-terminal protein Kinase (JNK) or Stress Activated Protein Kinase (SAPK). MATERIAL AND METHODS: CD34+ and CD34- fractions of four human normal bone marrow and unfractionated bone marrow samples were investigated. Immunoblottings, immunoenzymatic and in vitro assays were performed. RESULTS: Shc protein isoforms were constitutively expressed in all the AML cases examined. Tyrosine-phosphorylation of p53/p46Shc isoforms were found in CD34+ but not in the majority of CD34- cases. p66Shc isoform was not tyrosine-phosphorylated in CD34-, and was tyrosine-phosphorylated only in some CD34+ cases. Expression and activation of ERK was constitutively present in the majority of AML patients analysed. JNK/SAPK was expressed but not activated in the AMLs examined. Activation occurred after treatment of the leukemic cells by anisomycin, etoposide, and cytarabine. ERK and JNK/SAPK activation were not detectable in the hematopoietic precursors of human normal bone-marrow. CONCLUSION: These data bear implications for the role of Shc-MAP kinase pathway in normal hemopoiesis and AML leukemogenesis.