Amandine Pradier

Human Bone Marrow Stromal Cells and Skin Fibroblasts Inhibit Natural Killer Cell Proliferation and Cytotoxic Activity

Mesenchymal stromal cells (MSCs) are potent immunomodulators that have successfully been used to circumvent various types of inflammations, including steroid-resistant graft-versus-host disease. Although initially believed to be restricted to multipotent MSCs, this immunoregulatory function is shared with differentiated cells from the mesenchymal lineage such as skin fibroblasts (SFs). Mesenchymal cell-induced immunoregulation is so potent that it may allow the reactivation of dormant malignancies, a fact that would preclude using such cells as therapeutic agents. Because NK cells are pivotal effectors controlling tumor cell containment we investigated the effect of allogenic MSCs and SFs on NK cell function in vitro. When NK cells were incubated with IL-15 and MSCs or SFs for 6 days, their proliferation and cytotoxic activity were significantly decreased compared to NK cells cultured with IL-15 alone or with human venous endothelial cells. Cytotoxic activity inhibition reached 86% when assayed on MHC-I+ allogenic primary hematopoietic blasts, and was associated with a significant decrease in cytolytic granule exocytosis and in perforin release. Stromal cell-mediated inhibition was effective only if cell–cell proximity was long lasting: when NK cells were activated with IL-15 in the absence of MSCs and assayed for cytotoxicity in their presence no inhibition occurred. MSC inhibition was ultimately mediated by a soluble factor generated upon incubation with NK cells activated by IL-15 or IL-2. The indoleamine 2,3 dioxygenase was activated in MSCs and SFs because L-kynurenine was detected in inhibitory supernatants, but its blockade did not restore NK cell functions. The profound inhibition of cytotoxic activity directed against allogenic hematopoietic blasts exerted by MSCs and SFs on NK cells may be a concern. Should this occur in vivo it may induce the inability of NK cells to control residual or dormant malignant diseases after infusion of therapeutic MSCs.

T-bet and Eomesodermin in NK Cell Development, Maturation, and Function.

Recent reports give insights into the role of the T-box transcription factors, T-bet and Eomesodermin (Eomes), in NK cell biology. In this mini-review, we recapitulate the initial reports that delineate T-bet and Eomes as master regulators of NK cell development, maturation, and function. We discuss how T-bet and Eomes expression is regulated during NK cell development and peripheral maturation. Furthermore, we summarize the current literature on the role of T-bet and Eomes in the transcriptional regulation of NK cell function and review possible effects of T-box transcription factor anomalies during aging, infection, cancer, and after hematopoietic stem cell transplantation. We discuss how the current data argue in favor of a model of T-bet and Eomes synergy in transcriptional regulation of NK cell function and identify T-box transcription factors as potential targets for therapeutic interventions.

Torque teno virus in patients undergoing allogeneic hematopoietic stem cell transplantation for hematological malignancies

Torque teno virus in patients undergoing allogeneic hematopoietic stem cell transplantation for hematological malignancies

NK Cell Functional Impairment after Allogeneic Hematopoietic Stem Cell Transplantation Is Associated with Reduced Levels of T-bet and Eomesodermin

NK cells play a major role in protection against tumor recurrence and infection after allogeneic hematopoietic stem cell transplantation (HSCT). It has been shown that NK cell function after HSCT is impaired, but underlying molecular mechanisms are not well-known. In this report we show that the level of T-bet and Eomesodermin (Eomes), two T-box transcription factors regulating lymphocyte effector functions, is strongly reduced in NK cells from HSCT recipients compared with healthy control subjects. Reduction of T-bet and Eomes expression appeared early and persisted for years after HSCT, affecting all peripheral blood NK cells independently of their differentiation status. Reduced T-bet levels in NK cells from allogeneic HSCT recipients significantly correlated with reduced perforin expression. Acute, but not chronic, graft-versus-host disease, as well as CMV reactivation, was associated with further downregulation of T-bet expression in NK cells. Lower levels of T-bet expression in NK cells were associated with less favorable outcome after HSCT as a result of increased nonrelapse mortality. Collectively, our results provide a possible molecular explanation for the previously reported functional exhaustion of NK cells after allogeneic HSCT and suggest an impact of the NK transcriptional machinery status on HSCT outcome.

Validation of SYBR Green based quantification assay for the detection of human Torque Teno virus titers from plasma

BackgroundQuantification of titers of ubiquitous viruses such as Torque teno virus (TTV) that do not cause clinical symptoms might be helpful in assessing the immune status of an individual. We hereby describe the validation of a SYBR Green-based TTV quantification method for plasma samples.MethodsPlasmids with TTV specific inserts were used for preparing standards and absolute quantification of TTV was performed using SYBR Green methodology. The method was assessed for its accuracy and precision (intra and inter-day) on four non-consecutive days. TTV was also quantified from plasma samples of 20 healthy volunteers and from 30 hematopoietic stem cell transplant (HSCT) recipients.ResultsThe assay was specific and showed satisfactory efficiency (82.2%, R2=0.99) with the limit of quantification defined as 100 copies per reaction. The assay had good precision (inter and intra-day coefficient of variation in cycle threshold (CT) < 4%) and accuracy (100 ± 10%) in the range of 100 to 1010 copies/reaction. We found TTV loads ranging from 2.5 – 4.07 log copies/mL of plasma with CT (mean ± SD) of 33.8 ± 1.77 in healthy individuals and 2.06 – 8.49 log copies/mL of plasma with CT (mean ± SD) of 24.3 ± 1.04 in HSCT recipients.ConclusionSYBR Green-based q-PCR assay combines simplicity with satisfactory sensitivity and may be suitable for monitoring the immune status of transplant recipients, where TTV loads over time may serve as a marker for immune reconstitution in human plasma samples.

NK Cell Isolation from Liver Biopsies: Phenotypic and Functional Analysis of Low Cell Numbers by Flow Cytometry

Natural killer (NK) cells are considered to play a critical role in liver disease. However, the available numbers of intrahepatic lymphocytes (IHL) derived from liver biopsies (LB) for ex vivo analysis of intrahepatic NK cells is very limited; and the isolation method may hamper not only yields and viability, but also phenotype and function of IHL. The aim of the present study was therefore to (1) refine and evaluate the cell yields and viability of a modified isolation protocol from standard size needle LB; and (2) to test the effects of mechanical dissociation and enzymatic tissue digestion, as well as the analysis of very low cell numbers, on the phenotype and function of intrahepatic NK cells. Peripheral blood mononuclear cells (PBMC) and IHL, freshly isolated from the peripheral blood, LB (n = 11) or partial liver resections (n = 5), were used for phenotypic analysis by flow cytometry. NK cell function, i.e., degranulation and cytokine production, was determined by staining of CD107a and intracellular IFN-γ following in vitro stimulation. The mean weight of the LB specimens was 9.1 mg, and a mean number of 7,364 IHL/mg were obtained with a viability of >90%. Exposure of IHL and PBMC to 0.5 mg/ml collagenase IV and 0.02 mg/ml DNase I for 30 min did affect neither the viability, NK cell function, nor the percentages of CD56+, NKp46+, and CD16+ NK cells, whereas the level of CD56 surface expression was reduced. The phenotype of LB-derived NK cells was reliably characterized by acquiring as few as 2,500 IHL per tube for flow cytometry. The functional assay of intrahepatic NK cells was miniaturized by culturing as few as 25,000 IHL in 25 μl (106/ml) using 96-well V-bottom plates with IL-2 and IL-12 overnight, followed by a 4 h stimulation with K562 cells at a NK:K562 ratio of 1:1. In summary, we report reliable phenotypic and functional analyses of small numbers of intrahepatic NK cells isolated from LB specimens providing us with a tool to better address the emerging role of human NK cell immunobiology in liver diseases.

Torque Teno Virus Load and Acute Rejection After Orthotopic Liver Transplantation

decreases from 2.5% of total body weight in young people to about 1.6% in the nonagenarian population, and several morphological changes with aging indicate that the liver cells in advanced old age are in a hyperfunctioning state, possibly trying to compensate for the decline in total cell number. However, it has been suggested that aging has a limited effect on liver functions but more on its response to extrahepatic factors, disease states or increased metabolic demands to which elderly people may have an impaired ability to respond. Atheromatosis usually affects the celiac trunk in old donors but, although this happens rarely, when atheroma occurs distally at the level of the right hepatic artery or at the bifurcation of the gastroduodenal and the common hepatic artery, the liver graft must be discarded for LT. The LT procedure is a scenario in which many donor risk factors may be involved. Thus, in our previous brief communication on octogenarian liver donors, we recommended the use of liver grafts with no age limit but in good preprocurement condition (hemodynamic stability, low doses of vasopressor drugs), short intensive care unit stay, good liver function tests, soft liver consistency, absence of hepatic artery atheromatosis, cold ischemia time less than 9 hours, and macrosteatosis less than 30%. From that time, we have continued applying the same criteria, but in recent years we have added other conditions such as implanting

Modulation of T-bet and Eomes during Maturation of Peripheral Blood NK Cells Does Not Depend on Licensing/Educating KIR.

Peripheral natural killer (NK) cells upregulate T-bet and downregulate Eomes, the key transcription factors regulating NK cell maturation and function during the last maturation steps toward terminally differentiated effector cells. During this process, NK cells acquire killer immunoglobulin-like receptors (KIR) and effector functions, such as cytotoxicity and target cell-induced cytokine production. Inhibitory KIR are pivotal in the control of effector functions, but whether they also modulate T-bet/Eomes expression is unknown. We have measured T-bet/Eomes levels, KIR expression, and effector functions of maturing CD94negCD56dimNK cells using CD57 as surface marker for maturation. Our cohort consisted of 23 healthy blood donors (HBD) homozygous for the KIR A haplotype that contains only inhibitory KIR2DL1 (ligand HLA-C2), KIR2DL3 (ligand HLA-C1), and KIR3DL1 (ligand HLA-Bw4). We confirm that during maturation of NK cells, the number of KIR increases, levels of T-bet/Eomes are modulated, and that cells acquire effector functions, such as cytotoxicity (CD107) and target cell-induced cytokine production (TNF-α). Because maturation was associated with the increase of the number of KIR as well as with the modulation of T-bet/Eomes, the number of KIR correlated with the extent of T-bet/Eomes modulation. However, whether the KIR were triggered by their cognate HLA ligands or not had no impact on T-bet and Eomes expression, indicating that modulation of T-box transcription factors during NK cell maturation does not depend on signals conveyed by KIR. We discuss the relevance of this finding in the context of models of NK cell maturation while cautioning that results obtained in a perhaps quite heterogeneous cohort of HBD are not necessarily conclusive.

Low molecular weight dextran sulfate binds to human myoblasts and improves their survival after transplantation in mice

Myoblast transplantation represents a promising therapeutic strategy in the treatment of several genetic muscular disorders including Duchenne muscular dystrophy. Nevertheless, such an approach is impaired by the rapid death, limited migration, and rejection of transplanted myoblasts by the host. Low molecular weight dextran sulfate (DXS), a sulfated polysaccharide, has been reported to act as a cytoprotectant for various cell types. Therefore, we investigated whether DXS could act as a “myoblast protectant” either in vitro or in vivo after transplantation in immunodeficient mice. In vitro, DXS bound human myoblasts in a dose dependent manner and significantly inhibited staurosporine-mediated apoptosis and necrosis. DXS pretreatment also protected human myoblasts from natural killer cell-mediated cytotoxicity. When human myoblasts engineered to express the renilla luciferase transgene were transplanted in immunodeficient mice, bioluminescence imaging analysis revealed that the proportion of surviving myoblasts 1 and 3 days after transplantation was two times higher when cells were preincubated with DXS compared to control (77.9 ± 10.1% vs. 39.4 ± 4.9%, p = 0.0009 and 38.1 ± 8.5% vs. 15.1 ± 3.4%, p = 0.01, respectively). Taken together, we provide evidence that DXS acts as a myoblast protectant in vitro and is able in vivo to prevent the early death of transplanted myoblasts.

Peripheral blood CD56(bright) NK cells respond to stem cell factor and adhere to its membrane-bound form after upregulation of c-kit.

CD56bright NK cells express receptors for IL‐2, IL‐7, IL‐15, and SCF. We found that human peripheral blood CD56bright NK cells responded to IL‐2, IL‐7, IL‐15 by phosphorylating STAT‐5, ERK, and Akt but did not respond to SCF. However, CD56bright NK cells in culture upregulated c‐kit transcription three to fourfold, which led to a steady increase in c‐kit and a concomitant acquisition of responsiveness to SCF. After 44 h, CD56bright NK cells had upregulated c‐kit approximately 20‐fold and phosphorylated ERK and Akt in response to SCF concentrations well below levels present in plasma. CD56bright NK cells cultured in IL‐15 maintained c‐kit transcription/expression at ex vivo levels and did not become responsive to SCF. Furthermore, SCF‐responsive, CD56brightc‐kithigh NK cells swiftly downregulated c‐kit and stopped responding to SCF after IL‐15 stimulation. However, commitment of CD56bright NK cells to a c‐kit‐negative, SCF‐unresponsive state did not occur, as after 5 days of culture, withdrawal of IL‐15 restored c‐kit to maximal levels and reestablished SCF‐responsiveness. CD56bright NK cells that had upregulated c‐kit firmly adhered to COS cells transfected with the membrane form of SCF. Furthermore, SCF signaling significantly increased the capacity of CD56bright NK cells to degranulate. Collectively, our data suggest that c‐kit on human CD56bright NK cells is a functional receptor that is downregulated in peripheral blood, possibly to render CD56bright NK cells unresponsive to the SCF therein.