Philippe Lewalle

A survey of fully haploidentical hematopoietic stem cell transplantation in adults with high-risk acute leukemia : a risk factor analysis of outcomes for patients in remission at transplantation

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is an alternative treatment to patients with high-risk acute leukemia lacking a human leukocyte antigen-matched donor. We analyzed 173 adults with acute myeloid leukemia (AML) and 93 with acute lymphoblastic leukemia (ALL) who received a haplo-HSCT in Europe. All grafts were T cell-depleted peripheral blood progenitor cells from a direct family or other related donor. At transplantation, there were 25 patients with AML in CR1 (complete remission 1), 61 in more than or equal to CR2, and 87 in nonremission, and 24 with ALL in CR1, 37 in more than or equal to CR2, and 32 in nonremission. Median follow-up was 47 months in AML and 29 months in the ALL groups. Engraftment was observed in 91% of the patients. Leukemia-free survival at 2 years was 48% plus or minus 10%, 21% plus or minus 5%, and 1% for patients with AML undergoing transplantation in CR1, more than or equal to CR2, and nonremission, and 13% plus or minus 7%, 30% plus or minus 8%, and 7% plus or minus 5% in ALL patients, respectively. In conclusion, haplo-HSCT can be an alternative option for the treatment of high-risk acute leukemia patients in remission, lacking a human leukocyte antigen-matched donor.

Human natural Treg microRNA signature: Role of microRNA‐31 and microRNA‐21 in FOXP3 expression

Treg are the main mediators of dominant tolerance. Their mechanisms of action and applications are subjects of considerable debate currently. However, a human microRNA (miR) Treg signature has not been described yet. We investigated human natural Treg and identified a signature composed of five miR (21, 31, 125a, 181c and 374). Among those, two were considerably under‐expressed (miR‐31 and miR‐125a). We identified a functional target sequence for miR‐31 in the 3′ untranslated region (3′ UTR) of FOXP3 mRNA. Using lentiviral transduction of fresh cord blood T cells, we demonstrated that miR‐31 and miR‐21 had an effect on FOXP3 expression levels. We showed that miR‐31 negatively regulates FOXP3 expression by binding directly to its potential target site in the 3′ UTR of FOXP3 mRNA. We next demonstrated that miR‐21 acted as a positive, though indirect, regulator of FOXP3 expression. Transduction of the remaining three miR had no direct effect on FOXP3 expression or on the phenotype and will remain the subject of future investigations. In conclusion, not only have we identified and validated a miR signature for human natural Treg, but also unveiled some of the mechanisms by which this signature was related to the control of FOXP3 expression in these cells.

Mesenchymal stromal cells promote or suppress the proliferation of T lymphocytes from cord blood and peripheral blood: the importance of low cell ratio and role of interleukin-6

BACKGROUND AIMS Mesenchymal stromal cells (MSC) have been shown to possess immunomodulatory functions and proposed as a tool for managing or preventing graft-versus-host disease (GvHD) as well as promoting clinical transplantation tolerance. We investigated the capacity of human bone marrow (BM) MSC to modulate the proliferation of T cells obtained from peripheral blood (PB) and umbilical cord blood (CB). We addressed the importance of the MSC:T-cell ratio, requirement for cell contact and impact of soluble factors on the MSC-mediated effects. We also analyzed whether regulatory T cells could be modulated by MSC in co-cultures. METHODS The effect of different MSC concentrations on T-cell proliferation induced by allogeneic, mitogenic or CD3/CD28 stimulation was analyzed using bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate-succinimidyl ester (CFDA-SE) labeling. The level of regulatory T cells was assessed using quantitative real-time polymerase chain reaction (PCR) and flow cytometry analysis. RESULTS MSC induced a dose- and contact-dependent inhibition of T-cell proliferation but lymphocytes from CB and PB were differentially affected. At low concentrations, MSC supported both CB and PB T-cell proliferation, rather than inhibiting their proliferation. This supportive effect was contact independent and soluble factors such interleukin-6 (IL-6) appeared to be involved. Interestingly, among the expanded T-cell population in both CB and PB, regulatory T cells were increased and were a part of the new cells promoted by MSC at low doses. CONCLUSIONS MSC represent an attractive tool for reducing the lymphocyte response by inhibiting T-cell activation and proliferation as well as promoting tolerance by maintaining and promoting the expansion of regulatory cells. Nevertheless, the dual ability of MSC to either sustain or suppress T-cell proliferation according to conditions should be considered in the context of clinical applications.

Circulating miR-150 and miR-342 in plasma are novel potential biomarkers for acute myeloid leukemia

BackgroundMicroRNAs (miRNAs) are small (19-22-nt) single-stranded noncoding RNA molecules whose deregulation of expression can contribute to human disease including the multistep processes of carcinogenesis in human. Circulating miRNAs are emerging biomarkers in many diseases and cancers such as type 2 diabetes, pulmonary disease, colorectal cancer, and gastric cancer among others; however, defining a plasma miRNA signature in acute myeloblastic leukemia (AML) that could serve as a biomarker for diagnosis or in the follow-up has not been done yet.MethodsTaqMan miRNA microarray was performed to identify deregulated miRNAs in the plasma of AML patients. Quantitative real-time RT-PCR was used to validate the results. Receiver-operator characteristic (ROC) curve analysis was conducted to evaluate the diagnostic accuracy of the highly and significantly identified deregulated miRNA(s) as potential candidate biomarker(s).ResultsThe plasma expression level of let-7d, miR-150, miR-339, and miR-342 was down-regulated whilst that of let-7b, and miR-523 was up-regulated in the AML group at diagnosis compared to healthy controls. ROC curve analyses revealed an AUC (the areas under the ROC curve) of 0.835 (95% CI: 0.7119– 0.9581; P<0.0001) and 0.8125 (95% CI: 0.6796–0.9454; P=0.0005) for miR-150, and miR-342 respectively. Combined ROC analyses using these 2 miRNAs revealed an elevated AUC of 0.86 (95% CI: 0.7819–0.94; P<0.0001) indicating the additive effect in the diagnostic value of these 2 miRNAs. QRT-PCR results showed that the expression level of these two miRs in complete remission AML patients resembled that of healthy controls.ConclusionsOur findings indicated that plasma miR-150 and miR-342 are novel important promising biomarkers in the diagnosis of AML. These novel and promising markers warrant validation in larger prospective studies.

Infusion of Mesenchymal Stromal Cells can Aid Hematopoietic Recovery Following Allogeneic Hematopoietic Stem Cell Myeloablative Transplant: A Pilot Study

Mesenchymal stromal cells (MSCs) are important in the support of hematopoiesis. In this pilot study, we evaluated the safety and efficiency of donor-expanded MSC infusion after allogeneic hematopoietic stem cell transplantation (HSCT) in six patients with poor hematopoietic recovery. MSCs were infused without HSC and without conditioning at a dose of 1 x 10(6)/kg weight. Two patients displayed rapid hematopoietic recovery (days 12 and 21), and four patients showed no response. The two patients who showed hematopoietic recovery were in first complete remission (CR1) compared to the other heavily pretreated patients. There were no toxic side effects linked to MSC infusion. One patient developed cytomegalovirus (CMV) reactivation 12 days following the MSC infusion and died from CMV disease. We found that infusion of MSCs without HSC co-infusion can restore medullary function in some patients with poor hematopoietic recovery. Our data suggest that patients with a less damaged stroma could benefit from this approach.

Donor lymphocyte infusions in adult haploidentical transplant: a dose finding study.

Summary:Haploidentical transplantation has become a clinical option for patients lacking a compatible donor. However, patients are still referred at advanced stages and are usually heavily pretreated. This results in a high risk of toxicity, relapses and infections. We therefore started a donor lymphocyte infusion (DLI) dose-finding protocol, to try to improve both relapse rate and immunity reconstitution. In all, 12 consecutive patients were investigated. All had a refractory, some progressive, disease. Conditioning consisted of TBI, melphalan, ATG, fludarabine and CSA pretransplant. In four rapidly progressive patients, Ara-C had to be given 1 week preconditioning. The graft was T- and B-cell depleted with a fixed reinfused CD3 dose of 5×104/kg. All patients engrafted before day 20. G-CSF was given from day 5 post-transplant and replaced with GM-CSF in the last three patients. Nonrelapse related mortality was 0/12 at 1 year. DLI were started at day 28 (3×104 CD3/kg) in the two first patients. This resulted in acute graft-versus-host disease (aGVHD) and chronic graft-versus-host disease (cGVHD) in both, but they did not relapse. The next dose was 1×104/kg monthly for 3 months. This was well tolerated with only one grade I GVHD. Given the high relapse rate, we escalated doses (1, 3 and 10×104/kg). This produced GVHD in all. We next moved, to GM-CSF and 1×104 CD3/kg monthly. Overall, 6/12 patients relapsed and received therapeutic DLI, starting at 1×105 CD3/kg with escalation every 2 weeks. We conclude that prophylactic DLI are feasible in adult haploidentical transplantation, without GVHD at a monthly dose of 1×104 CD3/kg. They result in faster CD4 recovery and a low rate of infections. The impact of GM-CSF remains to be further investigated. This scheme seems ideal for patients transplanted early in the course of their disease. In very bad prognosis patients, it remains insufficient to rapidly induce a GVL effect. Escalated doses are feasible but the price is aGVHD. Therapeutic DLI can be given at higher doses, depending on the time post-transplant. Haploidentical transplantation with low-dose DLI is a safe procedure that should be considered in all patients needing a transplant, but lacking a matched donor, early in the course of the disease.

Freezing of dendritic cells, generated from cryopreserved leukaphereses, does not influence their ability to induce antigen-specific immune responses or functionally react to maturation stimuli

It is of practical clinical importance to be able to reinfuse into patients dendritic cells which have been previously frozen in aliquots. However, there are few studies comparing the function of fresh and frozen dendritic cells. We therefore decided to perform a systematic immunophenotypic and functional comparison of fresh and frozen dendritic cells. We chose to assess functional properties using proliferation tests and evaluating the preservation of specific antigens presentation in the context of MHC class II. Dendritic cells, generated from leukaphereses of normal volunteers, were loaded with proteins by a 2-h incubation at a protein concentration of 50 microg/ml, and were thereafter used fresh or after freeze-thawing in an IFNgamma Elispot assay. The IFNgamma release from antigen specific T cells was not affected by liquid nitrogen storage of pulsed immature dendritic cells. In the same way, the storage did not alter their stimulatory properties for antigen specific autologous T cells or for allogeneic CD8+ T lymphocytes in a proliferation assay. We also showed that freezing non-pulsed immature dendritic cells did not alter their capacity to capture, process and generate antigen-specific reactions once thawed, nor did it impair their capacity to acquire fully mature characteristics using CD40L and IFNgamma, with respect to immunophenotype and bioactive IL-12 secretion.

Lentiviral-mediated gene delivery in human monocyte-derived dendritic cells: Optimized design and procedures for highly efficient transduction compatible with clinical constraints

Gene delivery to dendritic cells (DCs) could represent a powerful method of inducing potent, long-lasting immunity. Although recent studies underline the intense interest in lentiviral vector–mediated monocyte-derived DC transduction, efficient gene transfer methods currently require high multiplicities of infection and are not compatible with clinical constraints. We have designed a strategy to optimize the efficiency and clinical relevance of this approach. Initially, using a third generation lentiviral vector expressing green fluorescent protein, we found that modifying the vector design, the DC precursor cell type, and the DC differentiation stage for transduction results in sustained transgene expression in 75–85% of immature DCs (transduction at a multiplicity of infection of 8). This high efficiency was reproducible among different donors irrespective of whether DCs were expanded from fresh or cryopreserved CD14+ precursors. We then developed procedures that bypass the need for highly concentrated lentiviral preparations and the addition of polybrene to achieve efficient transduction. DCs transduced under these conditions retain their immature phenotype and immunostimulatory potential in both autologous and allogeneic settings. Furthermore, genetically modified DCs maintain their ability to respond to maturation signals and secrete bioactive IL-12, indicating that they are fully functional. Finally, the level of transgene expression is preserved in the therapeutically relevant mature DCs, demonstrating that there is neither promoter-silencing nor loss of transduced cells during maturation. The novel approach described should advance lentiviral-mediated monocyte-derived DC transduction towards a clinical reality.

Preemptive management of Epstein-Barr virus reactivation after hematopoietic stem-cell transplantation.

Background. Epstein-Barr virus (EBV) reactivation after hematopoietic stem-cell transplantation can lead to posttransplant lymphoproliferative disease (PTLD), which carries a high mortality rate. Among therapeutic and prophylactic options being developed, B-cell depletion with monoclonal antibodies is encouraging. Because viral load after transplantation is correlated with PTLD occurrence, we developed a preemptive attitude based on polymerase chain reaction (PCR)-guided rituximab administration. Methods. We monitored 115 transplant patients with a quantitative PCR for EBV DNA performed on whole-blood samples. Criteria for treatment initiation were a single PCR above 40,000 DNA genome copies per milliliter (gCop/mL) or two rising values above 10,000 gCop/mL. Weekly rituximab infusion at the dose of 375 mg/m2 was administered until negative PCR results were available. We evaluated the incidence of EBV reactivation and PTLD development. Results. Nineteen patients (16.5%) met the criteria for treatment. Incidence of reactivation was the same in high-risk and standard-risk patients (12 vs. 7, P=0.38). One patient developed PTLD after discontinuation of therapy due to a serious adverse event. No other serious adverse events were noticed. Viral load disappeared after a median of three cycles of therapy, and weekly monitoring allowed prompt intervention. No PTLD-related death was observed, all-cause mortality in the treated population was 68%. Conclusions. Our PCR-guided and rituximab-based preemptive approach to avoid PTLD after allogeneic hematopoietic stem-cell transplantation is feasible but probably overtreated patients. Prospective trials are strongly needed, they should use uniform PCR techniques and consider higher threshold values for treatment initiation.

MicroRNA Profile of Circulating CD4-positive Regulatory T Cells in Human Adults and Impact of Differentially Expressed MicroRNAs on Expression of Two Genes Essential to Their Function

Background: Regulatory T cells are a subset of T cells with immunosuppressive properties, crucial for immune tolerance, which are also associated with cancer development. Results: The human circulating CD4+ Treg microRNA signature was identified. Conclusion: Differentially expressed microRNAs from the Treg miR signature directly and indirectly regulate crucial Treg genes (FOXP3 and CTLA-4). Significance: Identifying novel regulatory mechanisms of crucial Treg genes expression provides better insight into their biology and offers potential new targets for immunomodulatory therapies. Regulatory T cells (Tregs) are characterized by a high expression of IL-2 receptor α chain (CD25) and of forkhead box P3 (FOXP3), the latter being essential for their development and function. Another major player in the regulatory function is the cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) that inhibits cytotoxic responses. However, the regulation of CTLA-4 expression remains less well explored. We therefore studied the microRNA signature of circulating CD4+ Tregs isolated from adult healthy donors and identified a signature composed of 15 differentially expressed microRNAs. Among those, miR-24, miR-145, and miR-210 were down-regulated in Tregs compared with controls and were found to have potential target sites in the 3′-UTR of FOXP3 and CTLA-4; miR-24 and miR-210 negatively regulated FOXP3 expression by directly binding to their two target sites in its 3′-UTR. On the other hand, miR-95, which is highly expressed in adult peripheral blood Tregs, positively regulated FOXP3 expression via an indirect mechanism yet to be identified. Finally, we showed that miR-145 negatively regulated CTLA-4 expression in human CD4+ adult peripheral blood Tregs by binding to its target site in CTLA-4 transcript 3′-UTR. To our knowledge, this is the first identification of a human adult peripheral blood CD4+ Treg microRNA signature. Moreover, unveiling one mechanism regulating CTLA-4 expression is novel and may lead to a better understanding of the regulation of this crucial gene.