Functional evidence for derivation of systemic histiocytic neoplasms from hematopoietic stem/progenitor cells.

Langerhans cell histiocytosis (LCH) and the non-LCH neoplasm Erdheim-Chester disease (ECD) are heterogeneous neoplastic disorders marked by infiltration of pathologic macrophage-, dendritic cell-, or monocyte-derived cells in tissues driven by recurrent mutations activating MAPK signaling. Although recent data indicate that at least a proportion of LCH and ECD patients have detectable activating kinase mutations in circulating hematopoietic cells and bone marrow-based hematopoietic progenitors, functional evidence of the cell of origin of histiocytosis from actual patient materials has long been elusive. Here, we provide evidence for mutations in MAPK signaling intermediates in CD34+ cells from patients with ECD and LCH/ECD, including detection of shared origin of LCH and acute myelomonocytic leukemia driven by TET2-mutant CD34+ cell progenitors in one patient. We also demonstrate functional self-renewal capacity for CD34+ cells to drive the development of histiocytosis in xenotransplantation assays in vivo. These data indicate that the cell of origin of at least a proportion of patients with systemic histiocytoses resides in hematopoietic progenitor cells prior to committed monocyte/macrophage or dendritic cell differentiation and provide the first example of a patient-derived xenotransplantation model for a human histiocytic neoplasm.

T cell-intrinsic S1PR1 regulates endogenous effector T-cell egress dynamics from lymph nodes during infection.

Viral clearance requires effector T-cell egress from the draining lymph node (dLN). The mechanisms that regulate the complex process of effector T-cell egress from the dLN after infection are poorly understood. Here, we visualized endogenous pathogen-specific effector T-cell migration within, and from, the dLN. We used an inducible mouse model with a temporally disrupted sphingosine-1-phosphate receptor-1 (S1PR1) gene specifically in endogenous effector T cells. Early after infection, WT and S1PR1(-/-) effector T cells localized exclusively within the paracortex. This localization in the paracortex by CD8 T cells was followed by intranodal migration by both WT and S1PR1(-/-) T cells to positions adjacent to both cortical and medullary lymphatic sinuses where the T cells exhibited intense probing behavior. However, in contrast to WT, S1PR1(-/-) effector T cells failed to enter the sinuses. We demonstrate that, even when LN retention signals such as CC chemokine receptor 7 (CCR7) are down-regulated, T cell intrinsic S1PR1 is the master regulator of effector T-cell emigration from the dLN.

Human CD90 identifies Th17/Tc17 T cell subsets that are depleted in HIV-infected patients.

By revisiting CD90, a GPI-anchored glycoprotein, we show that CD90 is expressed by a subset of CD4(+) and CD8(+) human T cells. CD4(+)CD90(+) cells share similarities with Th17 cells because they express the Th17-specific transcription factor RORC2 and produce IL-17A. CD4(+)CD90(+) cells are activated memory T cells that express the gut mucosal markers CCR6, CD161, and the α(4) and ß(7) integrins. Compared with CD90-depleted CCR6(+) memory Th17 cells, CD4(+)CD90(+) cells express higher levels of IL-22 and proinflammatory cytokines (IL-6, TNF-α and GM-CSF), but they produce lower levels of IL-21 and no IL-9. Analyses of CD8(+)CD90(+) cells reveal that they express RORC2 and are able to produce higher levels of IL-17A, IL-22, and CCL20 compared with CD90-depleted CD8(+) cells. These data show that CD90 identifies Th17 and Tc17 cells with a peculiar cytokine profile. Studies of circulating CD90(+) cells in HIV patients show that CD90(+) cells are decreased with an imbalance of the CD4(+)CD90(+)/regulatory T cell ratio in nontreated patients compared with treated patients and healthy donors. Overall, human CD90 identifies a subset of Th17 and Tc17 cells within CD4(+) and CD8(+) T cells, respectively, which are depleted during HIV infection.

A phase I trial of adeno-associated virus serotype 1-γ-sarcoglycan gene therapy for limb girdle muscular dystrophy type 2C.

γ-Sarcoglycanopathy or limb girdle muscular dystrophy type 2C is an untreatable disease caused by autosomal recessively inherited mutations of the γ-sarcoglycan gene. Nine non-ambulatory patients (two males, seven females, mean age 27 years; range 16-38 years) with del525T homozygous mutation of the γ-sarcoglycan gene and no γ-sarcoglycan immunostaining on muscle biopsy were divided into three equal groups to receive three escalating doses of an adeno-associated virus serotype 1 vector expressing the human γ-sarcoglycan gene under the control of the desmin promoter, by local injection into the extensor carpi radialis muscle. The first group received a single injection of 3 × 10(9) viral genomes in 100 µl, the second group received a single injection of 1.5 × 10(10) viral genomes in 100 µl, and the third group received three simultaneous 100-µl injections at the same site, delivering a total dose of 4.5 × 10(10) viral genomes. No serious adverse effects occurred during 6 months of follow-up. All nine patients became adeno-associated virus serotype 1 seropositive and one developed a cytotoxic response to the adeno-associated virus serotype 1 capsid. Thirty days later, immunohistochemical analysis of injected-muscle biopsy specimens showed γ-sarcoglycan expression in all three patients who received the highest dose (4.7-10.5% positively stained fibres), while real-time polymerase chain reaction detected γ-sarcoglycan messenger RNA. In one patient, γ-sarcoglycan protein was detected by western blot. For two other patients who received the low and intermediate doses, discrete levels of γ-sarcoglycan expression (<1% positively stained fibres) were also detectable. Expression of γ-sarcoglycan protein can be induced in patients with limb girdle muscular dystrophy type 2C by adeno-associated virus serotype 1 gene transfer, with no serious adverse effects.

Defining biomarkers in oral cancer according to smoking and drinking status.

Introduction: Oral Squamous Cell Carcinomas (OSCC) are mostly related to tobacco consumption eventually associated to alcohol (Smoker/Drinker patients: SD), but 25-30% of the patients have no identified risk factors (Non-Smoker/Non-Drinker patients: NSND). We hypothesized that these patients have distinguishable immune profiles that could be useful for prognosis. Materials and Methods: Cells present in immune tumor microenvironment (TME) and blood from 87 OSCC HPV-negative patients were analyzed using a multiparameter flow cytometry assay, in a prospective case-control study. Cytokine levels in tumor supernatants and blood were determined by a cytometric bead array (CBA) assay. Results: Normal gingiva and blood from healthy donors (HD) were used as controls. A significant increase of granulocytes (p<0.05 for blood), of monocytes-macrophages (p<0.01 for blood) and of CD4+ T cells expressing CD45RO and CCR6 (p<0.001 for blood; p<0.0001 for TME) as well as higher levels of IL-6 (p<0.01 for sera, p<0.05 for tumor supernatant) were observed in SD patients as compared to NSND OSCC patients and HD. High percentages of CD4+ T cells expressing CD45RO and CCR6 cells in tumor tissue (p=0.05) and blood (p=0.05) of SD OSCC patients were also associated with a poorer prognosis while a high percentage of regulatory T cells (Treg) in tumor tissue was associated with a more favorable prognostic factor (p=0.05). Also, a higher percentage of blood CD8+ T lymphocytes among CD45+ cells in NSND patients was associated with a better disease-free survival (p=0.004). Conclusion: Granulocytes, monocytes-macrophages, and CD4+ T cells expressing CD45RO and CCR6 in blood and TME as well as serum IL-6 can therefore distinguish OSCC SD and NSND patients. Quantifying the proportion of CD4+ T cells expressing CD45RO and CCR6 and of Treg in SD patients and CD8+ T cells in NSND patients could help defining the prognostic of OSCC patients.

Tertiary Lymphoid Structure-B Cells Narrow Regulatory T Cells Impact in Lung Cancer Patients.

The presence of tertiary lymphoid structures (TLS) in the tumor microenvironment is associated with better clinical outcome in many cancers. In non-small cell lung cancer (NSCLC), we have previously showed that a high density of B cells within TLS (TLS-B cells) is positively correlated with tumor antigen-specific antibody responses and increased intratumor CD4+ T cell clonality. Here, we investigated the relationship between the presence of TLS-B cells and CD4+ T cell profile in NSCLC patients. The expression of immune-related genes and proteins on B cells and CD4+ T cells was analyzed according to their relationship to TLS-B density in a prospective cohort of 56 NSCLC patients. We observed that tumor-infiltrating T cells showed marked differences according to TLS-B cell presence, with higher percentages of naïve, central-memory, and activated CD4+ T cells and lower percentages of both immune checkpoint (ICP)-expressing CD4+ T cells and regulatory T cells (Tregs) in the TLS-Bhigh tumors. A retrospective study of 538 untreated NSCLC patients showed that high TLS-B cell density was even able to counterbalance the deleterious impact of high Treg density on patient survival, and that TLS-Bhigh Treglow patients had the best clinical outcomes. Overall, the correlation between the density of TLS-Bhigh tumors with early differentiated, activated and non-regulatory CD4+ T cell cells suggest that B cells may play a central role in determining protective T cell responses in NSCLC patients.

Comparative binding and uptake of liposomes decorated with mannose oligosaccharides by cells expressing the mannose receptor or DC-SIGN.

Mannose Receptor (MR) and DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) are two mannose-specific targets for antigens carried by liposomes but DC-SIGN is more specific of DCs. Here, DC targeting is addressed by using DPPC/DOPE liposomes decorated with a series of diether lipids with a polar head of either a mannose (Man), tri-antenna of α-d-mannopyranoside (Tri-Man), [Manα1-3(Manα1-6)Man] (Man-tri), pseudo-Man4 (PMan4) or pseudo-Man5 (PMan5). Liposomes decorated with Man-Tri show the highest binding and internalization in cells expressing DC-SIGN and in human monocytes-derived DCs. Conversely, cells expressing MR bind and take up Tri-Man liposomes 3-fold higher than Man-tri liposomes. Comparatively, liposomes decorated with PMan4 and PMan5 do not show any advantages. Overall, the results indicate that liposomes decorated with Man-tri residues are more selective toward DCs than those with Tri-Man thanks to better recognition by DC-SIGN.

Cigarette smoking induces human CCR6+Th17 lymphocytes senescence and VEGF-A secretion.

Chronic exposure to environmental pollutants is often associated with systemic inflammation. As such, cigarette smoking contributes to inflammation and lung diseases by inducing senescence of pulmonary cells such as pneumocytes, fibroblasts, and endothelial cells. Yet, how smoking worsens evolution of chronic inflammatory disorders associated with Th17 lymphocytes, such as rheumatoid arthritis, psoriasis, Crohn's disease, and multiple sclerosis, is largely unknown. Results from human studies show an increase in inflammatory CD4+ Th17 lymphocytes at blood- and pulmonary level in smokers. The aim of the study was to evaluate the sensitivity of CD4+ Th17 lymphocytes to cigarette smoke-induced senescence. Mucosa-homing CCR6+ Th17- were compared to CCR6neg -and regulatory T peripheral lymphocytes after exposure to cigarette smoke extract (CSE). Senescence sensitivity of CSE-exposed cells was assessed by determination of various senescence biomarkers (ß-galactosidase activity, p16Ink4a- and p21 expression) and cytokines production. CCR6+ Th17 cells showed a higher sensitivity to CSE-induced senescence compared to controls, which is associated to oxidative stress and higher VEGFα secretion. Pharmacological targeting of ROS- and ERK1/2 signalling pathways prevented CSE-induced senescence of CCR6+Th17 lymphocytes as well as VEGFα secretion. Altogether, these results identify mechanisms by which pro-oxidant environmental pollutants contribute to pro-angiogenic and pathogenic CCR6+Th17 cells, therefore potential targets for therapeutic purposes.

Massive expansion of regulatory T-cells following interleukin 2 treatment during a phase I-II dendritic cell-based immunotherapy of metastatic renal cancer.

Cytotoxic chemotherapy is ineffective in metastatic renal cancer. However, systemic administration of interleukin 2 (IL-2) or infusion of dendritic cells (DCs) loaded with tumor extracts can lead to some response rates with concomitant survival improvements. We report the results of a phase I-II pilot study combining DCs and IL-2 where six patients were included. DCs were derived from bone marrow CD34+ cells and loaded with autologous tumor extracts. CD34-DC vaccines were infused subcutaneously at day 45, 52, 59, 90 and 120 following surgery in combination with IL-2, that was subsequently administrated after the 3rd and 4th DC vaccinations. Preparation of tumor extracts and CD34-DCs were satisfactory in all patients but one. Due to rapid tumor progression, one patient was excluded before vaccination. In the 4 remaining patients, two received 3 vaccinations, while the 2 others received 5 vaccinations and the full IL-2 treatment. No adverse effect due to the vaccinations was observed. A specific immune response against autologous tumor cells was observed in the 2 patients who completed the treatment. Interestingly, these 2 patients had a more prolonged survival than the patients receiving 3 vaccinations. Importantly, a transient and massive increase of circulating natural regulatory T-cells (nTregs) was evidenced in 3 patients following IL-2 administration. Overall, the use of CD34-DC vaccines is feasible, safe and non-toxic. A specific anti-tumor immune response can be detected. However, our data highlights that IL-2 is a potent inducer of nTregs in vivo and as such may have a negative impact on cancer immunotherapy.

A first experience of transduction for differentiated HepaRG cells using lentiviral technology.

Currently, there is a lack of systems for studying the role of hepatitis B viral proteins, such as HBeAg and HBcAg, on liver injury. It is necessary to develop an original tool in order to clarify the role of these viral proteins in hepatic stellate cell activation, and to understand the molecular mechanisms of liver injury. HepaRG are the most reliable hepatocyte-like cells for studying liver functions or disorders. In this paper, we demonstrate that the transduction of differentiated HepaRG (dHepaRG) cells can be performed successfully using lentiviral particles. The production of a functional Green Fluorescent Protein (GFP) assessed by Fluorescence Activated Cell Sorting and fluorescence microscopy is up to 16% of GFP positive cells using a multiplicity of infection (MOI) of 2.4. We demonstrate that this technology can allow the stable expression of GFP during the long lifecycle of the cell (up to four weeks after the cell's passage). With this innovative tool, we aim to express viral proteins such as HBeAg or HBcAg in dHepaRG cells. The preliminary results of this work shows that HBeAg can be efficiently produced in dHepaRG cells and that increased MOI allows a better production of this protein. Our future objective will be to study the role of HBc and HBe proteins on the induction of hepatic fibrosis.

Clinical-grade preparation of human natural regulatory T-cells encoding the thymidine kinase suicide gene as a safety gene.

BACKGROUND: Human CD4+CD25+FOXP3+ natural regulatory T-cells (nTreg) have a great therapeutic potential for the induction of tolerance in allo-transplanted patients or for the control of severe auto-immune diseases. However, clinical-grade production of nTreg remains difficult to achieve because of the absence of a truly specific surface marker and of their low frequency that implies a need for their ex vivo expansion. Furthermore, safety issues should be taken into consideration due to the risk of either uncontrolled nTreg-induced immunosuppression or uncontrolled proliferation of autoreactive contaminating T-cells particularly in an auto-immune context. METHODS: We compared different clinical-grade conditions for immuno-magnetic selection and ex vivo expansion of nTreg. For safety, expanded cells were genetically modified with retroviral vectors co-expressing human CD90 and HSV1 thymidine kinase. The CD90 surface marker and thymidine kinase allow for selection and elimination of transduced cells by ganciclovir, respectively. RESULTS: We showed that (i) nTreg could be enriched in a one step using CD25 microbeads, were functionally suppressive and mainly FOXP3+; (ii) using anti-CD28- and anti-CD3-coated beads, interleukin-2 and rapamycin, nTreg were expanded 150-200-fold after 3 weeks. Under these clinical-grade conditions, they remained suppressive, and no major alteration of the TCR repertoire was observed; (iii) after efficient retroviral transduction and CD90 selection, nTreg maintained their suppressive activity; (iv) transduced nTreg could be eliminated by ganciclovir upon activation. CONCLUSIONS: The efficient procedure reported here for the preparation of nTreg, whose safety has been ensured, is now applicable for further clinical trials.

CD4 regulation in human lymphoid non-T-cells: a role for the silencer element.

In humans, the CD4 molecule is expressed on a subset of T-cells and at various levels on myeloid and lymphoid cells. The mechanisms regulating human CD4 gene expression are yet poorly understood. We speculated that the CD4 silencer, which operates in CD8+ T-cells to repress CD4 expression, could be responsible for CD4 repression in human lymphoid non-T-cells. To test this possibility, we used lentiviral vectors carrying CD4 regulatory sequences, with or without the silencer element, to express an eGFP reporter gene. We observed that (i) in the absence of the silencer element, eGFP expression was detected in CD34+-derived B- and NK-cells that otherwise lacked endogenous CD4 mRNA, indicating active repression of the CD4 regulatory sequences and (ii) the addition of the CD4 silencer could repress eGFP expression in these same cells, as well as in human B-cells generated in vivo in NOD/SCID mice. Collectively, our results suggest that beyond its well-characterized function in T-cells, the CD4 silencer also regulates CD4 gene expression in human lymphoid non-T-cells.

Novel mode of action of c-kit tyrosine kinase inhibitors leading to NK cell-dependent antitumor effects.

Mutant isoforms of the KIT or PDGF receptors expressed by gastrointestinal stromal tumors (GISTs) are considered the therapeutic targets for STI571 (imatinib mesylate; Gleevec), a specific inhibitor of these tyrosine kinase receptors. Case reports of clinical efficacy of Gleevec in GISTs lacking the typical receptor mutations prompted a search for an alternate mode of action. Here we show that Gleevec can act on host DCs to promote NK cell activation. DC-mediated NK cell activation was triggered in vitro and in vivo by treatment of DCs with Gleevec as well as by a loss-of-function mutation of KIT. Therefore, tumors that are refractory to the antiproliferative effects of Gleevec in vitro responded to Gleevec in vivo in an NK cell-dependent manner. Longitudinal studies of Gleevec-treated GIST patients revealed a therapy-induced increase in IFN-gamma production by NK cells, correlating with an enhanced antitumor response. These data point to a novel mode of antitumor action for Gleevec.

Characteristics of hybrid cells obtained by dendritic cell/tumour cell fusion in a T-47D breast cancer cell line model indicate their potential as anti-tumour vaccines.

Many strategies have been proposed to circumvent cancer development or prevent its growth. One of the promising strategies is to direct the immune response toward tumour antigens. This can be achieved by loading dendritic cells, the most potent antigen presenting cells, with tumour antigens. Fusion of dendritic cells (DC) with tumour cells is an attractive way to load the DC with all tumour antigens regardless of their immunogenicity status and the fact that they have, or not, been identified. The aim of our study was to characterise the immunophenotype of fused cells, monitor the evolution of the fusion interface and the distribution of surface antigens over time and assess for their maturation status and functionality in vitro. We used polyethylene glycol to fuse DC with Her2/neu positive breast cancer cell line T-47D. We demonstrate that false positive events accounted in flow cytometry can be identified using confocal microscopy to avoid an overestimation of fusion efficiency and to distinguish clearly hybrid cells from aggregated or phagocytosed cells. We used imaging means to demonstrate the conservation of presentation molecules (MHC II, CD1a), co-stimulatory molecules (CD40, CD80, CD86), as well as tumour antigens (Her2/neu, cytokeratins) in optimised conditions. Fused cells were only recognisable for 48 h as assessed by membrane staining and membranous antigen distribution. Fusion was necessary for their maturation to be accompanied by functional activity such as secretion of cytokines and perforin. These results suggest that hybrid cells generated by the fusion of DC and tumour cells can be easily identified and characterised using imaging techniques, and that, regarding functionality and cytokine secretion, they appear to be good candidates for anti-tumour therapies namely in breast cancer.

Regulatory T Cell Therapy for Uveitis: A New Promising Challenge.

Uveitis is a sight-threatening primary intraocular inflammation of various origins in mainly young and active patients. Due to the absence of biomarkers in most of the cases, the current treatment of noninfectious entities remains nonspecific, using corticosteroids, conventional immunosuppressors, and more recently biological agents. Identification of regulatory T cells in different models of autoimmune uveitis together with the evaluation of this important subpopulation in different entities paved the way for new therapeutic strategies, in addition to exclusive pharmaceutical approaches. Upregulation of regulatory T cells induced by biological agents has been recently highlighted. Development of cell therapy in autoimmune diseases is at its stammering needing more experimental data and robust clinical trials to demonstrate safety and efficacy before larger developments. Specific or polyclonal Tregs may be used, but it is of utmost importance to determine the method of selection, the level of activation, and the route of administration. Mastering immune cell therapy remains a challenging goal in patients with autoimmune diseases, but it may significantly enlarge our therapeutic possibilities in severe and refractory situations.

Effect of intracoronary administration of AAV1/SERCA2a on ventricular remodelling in patients with advanced systolic heart failure: results from the AGENT-HF randomized phase 2 trial.

AIMS: Restoration of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2a) activity through gene transfer improved cardiac function in experimental and pilot studies in humans with heart failure. The AGENT-HF (NCT01966887) trial investigated the impact of adeno-associated virus (AAV1)/SERCA2a on ventricular remodelling using multimodality non-invasive cardiac imaging. METHODS AND RESULTS: AGENT-HF was a single centre, randomized, double-blind, placebo-controlled trial in adult patients with NYHA class III-IV ischaemic or non-ischaemic heart failure and left ventricular ejection fraction ≤35%. Eligible patients were randomized to receive a single intracoronary infusion of either 1 × 1013 DNase-resistant particles of AAV1/SERCA2a or placebo. The primary endpoint was change in left ventricular end-systolic volume (LVESV), measured by cardiac computed tomography at 6 month follow-up. We planned to include 40 patients but the trial was terminated prematurely as the sponsor suspended further enrolment following neutral results of the CUPID-2 outcome trial. At the time of termination, nine patients were randomized with five patients infused with AAV1/SERCA2a and four with placebo. At 6 months, LVESV was increased in both groups compared with baseline: median (interquartile range) in AAV1/SERCA2a vs. placebo: 13 (13;14) mL vs. 3.5 (-36;36) mL, P = 0.74, with a mean difference between groups of 11.4 mL in favour of placebo. No safety issues were noted. CONCLUSION: AGENT-HF failed to demonstrate any improvement in ventricular remodelling in response to AAV1/SERCA2a at the dose studied. However, because of premature termination, the study was underpowered to demonstrate an effect of AAV1/SERCA2a and these data should be interpreted with caution.

Engineered mesenchymal stem cells as vectors in a suicide gene therapy against preclinical murine models for solid tumors.

Gene-directed enzyme pro-drug therapy (GDEPT) consists of expressing, in tumor cells, a suicide gene which converts a pro-drug into cytotoxic metabolites, in situ. In a previous work, we demonstrated that the combination of the suicide gene CYP2B6TM-RED (a fusion of a triple mutant of CYP2B6 with NADPH cytochrome P450 reductase) and cyclophosphamide (CPA) constituted a powerful treatment for solid tumors. In this work, we investigated the use of mesenchymal stem cells (MSCs) as cellular vehicles for the delivery of our suicide gene. MSCs were genetically engineered ex-vivo to stably express CYP2B6TM-RED. Ex vivo and in vivo investigations showed that MSCs expressing CYP2B6TM-RED were able 1) to bioactivate CPA and produce local cytotoxic metabolites in tumor sites and 2) to destroy neighboring tumor cells through a bystander effect. Intratumoral injections of CYP2B6TM-RED-MSCs and CPA completely eradicated tumors in 33% of mice without recurrence after 6months. Rechallenge experiments demonstrated an efficient immune response. These data suggest that MSCs expressing CYP2B6TM-RED with CPA could represent a promising treatment for solid tumors to test in future clinical trials.