Mame Massar Dieng

Use of immunoglobulins in the prevention of GvHD in a xenogeneic NOD/SCID/γc− mouse model

The efficacy of IVIG in preventing GvHD has not been definitely demonstrated clinically. Using a xenogeneic model of GvHD in NOD/SCID/γc− (NSG) mice, we showed that weekly administration of IVIG significantly reduced the incidence and associated mortality of GvHD to a degree similar to CsA. Unlike CsA and OKT3, IVIG were not associated with inhibition of human T-cell proliferation in mice. Instead, IVIG significantly inhibited the secretion of human IL-17, IL-2, IFN-γ and IL-15 suggesting that IVIG prevented GvHD by immunomodulation. Furthermore, the pattern of modification of the human cytokine storm differed from that observed with CsA and OKT3. Finally, in a humanized mouse model of immune reconstitution, in which NSG mice were engrafted with human CD34+ stem cells, IVIG transiently inhibited B-cell reconstitution, whereas peripheral T-cell reconstitution and thymopoiesis were unaffected. Together these in vivo data raise debate related to the appropriateness of IVIG in GvHD prophylaxis. In addition, this model provides an opportunity to further elucidate the precise mechanism(s) by which IVIG inhibit GvHD.

Efficient BST2 antagonism by Vpu is critical for early HIV-1 dissemination in humanized mice

BackgroundVpu is a multifunctional accessory protein that enhances the release of HIV-1 by counteracting the entrapment of nascent virions on infected cell surface mediated by BST2/Tetherin. Vpu-mediated BST2 antagonism involves physical association with BST2 and subsequent mislocalization of the restriction factor to intracellular compartments followed by SCF(β-TrCP) E3 ligase-dependent lysosomal degradation. Apart from BST2 antagonism, Vpu also induces down regulation of several immune molecules, including CD4 and SLAMF6/NTB-A, to evade host immune responses and promote viral dissemination. However, it should be noted that the multiple functions of Vpu have been studied in cell-based assays, and thus it remains unclear how Vpu influences the dynamic of HIV-1 infection in in vivo conditions.ResultsUsing a humanized mouse model of acute infection as well as CCR5-tropic HIV-1 that lack Vpu or encode WT Vpu or Vpu with mutations in the β-TrCP binding domain, we provide evidence that Vpu-mediated BST2 antagonism plays a crucial role in establishing early plasma viremia and viral dissemination. Interestingly, we also find that efficient HIV-1 release and dissemination are directly related to functional strength of Vpu in antagonizing BST2. Thus, reduced antagonism of BST2 due to β-TrCP binding domain mutations results in decreased plasma viremia and frequency of infected T cells, highlighting the importance of Vpu-mediated β-TrCP-dependent BST-2 degradation for optimal initial viral propagation.ConclusionsOverall, our findings suggest that BST2 antagonism by Vpu is critical for efficient early viral expansion and dissemination during acute infection and as such is likely to confer HIV-1 increased transmission fitness.

Therapeutic Efficacy of Cord Blood-Derived Mesenchymal Stromal Cells for the Prevention of Acute Graft-Versus-Host Disease in a Xenogenic Mouse Model

Human mesenchymal stromal cells (MSCs) have been successfully utilized for the treatment of refractory graft-versus-host disease (GvHD). Despite the large number of in vitro and in vivo models developed for clarifying their immunomodulatory properties, the mechanism of action of MSCs remains elusive and their efficacy controversial. Here, we tested the ability of cord blood-derived MSCs to alleviate the symptoms of GvHD induced by the injection of human peripheral blood mononuclear cells into NOD/SCID/γc(-) mice. In this in vivo xeno-GvHD model, we demonstrate that a single MSC injection is able to inhibit GvHD in terms of clinical signs and related mortality. We also show that in this model MSCs act by both immunomodulating T-cells and fostering recovery after irradiation. The translational impact of these findings could provide a reliable preclinical model for studying the efficacy, dosage, and time of administration of human MSCs for the prevention of acute GvHD.

Human mesenchymal stromal cell-secreted lactate induces M2-macrophage differentiation by metabolic reprogramming

Human mesenchymal stromal cells (MSC) have been shown to dampen immune response and promote tissue repair, but the underlying mechanisms are still under investigation. Herein, we demonstrate that umbilical cord-derived MSC (UC-MSC) alter the phenotype and function of monocyte-derived dendritic cells (DC) through lactate-mediated metabolic reprogramming. UC-MSC can secrete large quantities of lactate and, when present during monocyte-to-DC differentiation, induce instead the acquisition of M2-macrophage features in terms of morphology, surface markers, migratory properties and antigen presentation capacity. Microarray expression profiling indicates that UC-MSC modify the expression of metabolic-related genes and induce a M2-macrophage expression signature. Importantly, monocyte-derived DC obtained in presence of UC-MSC, polarize naïve allogeneic CD4+ T-cells into Th2 cells. Treatment of UC-MSC with an inhibitor of lactate dehydrogenase strongly decreases lactate concentration in culture supernatant and abrogates the effect on monocyte-to-DC differentiation. Metabolic analysis further revealed that UC-MSC decrease oxidative phosphorylation in differentiating monocytes while strongly increasing the spare respiratory capacity proportional to the amount of secreted lactate. Because both MSC and monocytes are recruited in vivo at the site of tissue damage and inflammation, we propose the local increase of lactate concentration induced by UC-MSC and the consequent enrichment in M2-macrophage generation as a mechanism to achieve immunomodulation.

Impact of storage temperature and processing delays on cord blood quality: discrepancy between functional in vitro and in vivo assays

BACKGROUND: Optimal conditions of cord blood (CB) storage, processing, cryopreservation, and thawing are critical for banking and transplantation. Nevertheless, standardized procedures are still awaited.

Cord-Blood-Derived Mesenchymal Stromal Cells Downmodulate CD4+ T-Cell Activation by Inducing IL-10-Producing Th1 Cells

The mechanisms by which mesenchymal stromal cells (MSCs) induce immunomodulation are still poorly understood. In the current work, we show by a combination of polymerase chain reaction (PCR) array, flow cytometry, and multiplex cytokine data analysis that during the inhibition of an alloantigen-driven CD4+ T-cell response, MSCs induce a fraction of CD4+ T-cells to coexpress interferon-γ (IFNγ) and interleukin-10 (IL-10). This CD4+ IFNγ+ IL-10+ cell population shares properties with recently described T-cells originating from switched Th1 cells that start producing IL-10 and acquire a regulatory function. Here we report that IL-10-producing Th1 cells accumulated with time during T-cell stimulation in the presence of MSCs. Moreover, MSCs caused stimulated T-cells to downregulate the IFNγ receptor (IFNγR) without affecting IL-10 receptor expression. Further, the inhibitory effect of MSCs could be reversed by an anti-IFNγR-blocking antibody, indicating that IFNγ is one of the major players in MSC-induced T-cell suppression. Stimulated (and, to a lesser extent, resting) CD4+ T-cells treated with MSCs were able to inhibit the proliferation of autologous CD4+ T-cells, demonstrating their acquired regulatory properties. Altogether, our results suggest that the generation of IL-10-producing Th1 cells is one of the mechanisms by which MSCs can downmodulate an immune response.

Human interferon-alpha increases the cytotoxic effect of CD56+cord blood-derived cytokine-induced killer cells on human B-acute lymphoblastic leukemia cell lines

BACKGROUND AIMS Cytokine-induced killer (CIK) cells may represent a promising immunotherapy for the treatment of children with relapsing B-lineage acute lymphoblastic leukemia (B-ALL) following hematopoietic stem cell transplantation (HSCT). Therefore, we investigated the possibility of combining adoptive immunotherapy with CIK cells and human interferon-alpha (hIFN-α) in order to potentiate the cytotoxicity of CIK cells against B-ALL. METHODS Cord blood- derived CIK (CB-CIK) cells were differentiated, stimulated with phosphate-buffered saline (PBS) or hIFN-α, and tested for cytotoxic activity. We tested the anti-leukemic and graft-versus-host disease (GvHD) effects of CB-CIK cells in a human xenograft NOD/SCID/γc(-) (NSG) mouse model. RESULTS Bulk CB-CIK cells showed very moderate cytotoxic activity while the subpopulation of CD56(+) CB-CIK cells showed significant cytotoxic activity against B-ALL cells. hIFN-α significantly augmented the cytotoxicity of CD56(+)CB-CIK cells in vitro and induced signal transducer and activator of transcription-1 (STAT1) phosphorylation. In addition, CD56(+)CB-CIK cells could delay mouse mortality significantly in vivo, and this effect was enhanced significantly by hIFN-α (P = 0.022). Furthermore, unlike CB mononuclear cells or peripheral blood mononuclear cells (PBMC), CD56(+)CB-CIK cells, alone or stimulated with hIFN-α, caused either no GvHD or mild GvHD, respectively, when injected into sublethally irradiated NSG mice. CONCLUSIONS CD56(+)CB-CIK cells are effective cytotoxic agents against human B-ALL cell lines in vitro and possess anti-leukemic activity that is potentiated by hIFN-α in an NSG mouse model in vivo. These pre-clinical data support the testing of this immunotherapeutic approach in the clinic for the treatment of B-ALL.

Implication of different effector mechanisms by cord blood–derived and peripheral blood–derived cytokine-induced killer cells to kill precursor B acute lymphoblastic leukemia cell lines

BACKGROUND AIMS Cytokine-induced killer (CIK) cells ex vivo-expanded from cord blood (CB) or peripheral blood (PB) have been shown to be cytotoxic against autologous and allogeneic tumor cells. We have previously shown that CD56(+) CIK cells (CD3(+)CD56(+) and CD3(-)CD56(+)) are capable of killing precursor B-cell acute lymphoblastic leukemia (B-ALL) cell lines. However, the lytic pathways used by CD56(+) PB and CB-CIK cells to kill B-ALL cell lines have not been studied. METHODS CB and PB-CIK cells were differentiated. CD56(+) CB- and PB-CIK cells were compared for expression of different phenotypic markers and for the lytic pathways used to kill B-ALL cell lines. RESULTS We found that cytotoxic granule proteins were expressed at higher levels in CD56(+) PB-CIK than in CD56(+) CB-CIK cells. However, CD56(+) CB-CIK cells expressed more tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) compared with CD56(+) PB-CIK cells. We observed that CD56(+) CB-CIK cells used both the NKG2D and TRAIL cytotoxic pathways and were more effective at killing REH cells than CD56(+) PB-CIK cells that used only the NKG2D pathway. In contrast, CD56(+) PB-CIK cells used both NKG2D and TRAIL pathways to kill NALM6 cells, whereas CD56(+) CB-CIK cells used only the NKG2D pathway. CONCLUSIONS Our results suggest that both the source of CIK and the type of B-ALL cell line have an impact on the intensity of the cytolytic activity and on the pathway used. These findings may have clinical implications with respect to optimizing therapeutic efficacy, which may be dependent on the source of the CIK cells and on the target tumor cells.

Cord blood-derived and peripheral blood-derived cytokine-induced killer cells are sensitive to Fas-mediated apoptosis.

Fas-mediated apoptosis is one of the mechanisms used by tumor cells to escape the cytotoxicity of tumor-infiltrating lymphocytes. It has been suggested that cytokine-induced killer (CIK) cells are resistant to Fas-mediated apoptosis, thereby rendering them more attractive for use in cellular immunotherapy. Unlike what was observed by others, here we show that CIK cells are sensitive to Fas-mediated apoptosis. We have observed an increase in Fas expression in the different CIK cell subpopulations (CD3(+)CD56(-), CD3(+)CD56(+), and CD3(-)CD56(+)) isolated from both cord blood (CB) and peripheral blood (PB). We also show that the bulk, as well as the CD3(+)CD56(-) and CD56(+) CB- and PB-CIK cell subpopulations were sensitive to Fas-mediated apoptosis induced by both CH11 and APO-1 antibodies, albeit with a weaker effect for the CH11 antibody on CB-CIK cells. In addition, in the presence of the APO-1 and CH11 inducers, Fas engagement inhibited the cytotoxic activity of CB- and PB-CIK cells. This new contradictory result may help explain the variable efficacy observed with CIK cells in the clinic.