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Lenalidomide is a potent anti-myeloma drug with immunomodulatory properties. It is increasingly used in a low-dose maintenance setting to prolong remission duration after standard treatment. Data on the in vivo effects of lenalidomide are scarce and sometimes different from the well-described in vitro effects. We therefore evaluated the numerical, phenotypical and functional impact of lenalidomide maintenance on several immune cell types in a cohort of seventeen homogeneously treated myeloma patients achieving a low residual myeloma burden after a bortezomib based-induction followed by autologous stem cell transplantation. Lenalidomide maintenance: 1) increased the fraction of naïve CD8+ T cells and several memory T-cell subsets, 2) reduced the numbers of terminal effector CD8+ T cells, 3) resulted in a higher expression of co-stimulatory molecules on resting T cells and of the inhibitory checkpoint molecules LAG-3 on CD4+ T cells and TIM-3 on CD4+ and CD8+ T cells, 4) reduced the number of TIGIT+ CD8+ T cells, 5) increased the number of regulatory T cells with a phenotype associated with strong suppressive capacity. Purified CD8+ T cells showed increased and more polyfunctional recall viral responses. However, PBMC responses were not enhanced during lenalidomide maintenance and CD4+ T-cell responses specific for the myeloma-associated antigen MAGE-C1 even tended to become lower. We conclude that lenalidomide maintenance after autologous stem cell transplantation has complex pleotropic effects on the immune environment. Immune interventions such as anti-myeloma vaccination should include measures to tackle an expanded inhibitory Treg compartment., SCOPUS: ar.j, info:eu-repo/semantics/published

Modulating the activity of tumor-infiltrating dendritic cells (TiDCs) provides opportunities for novel cancer interventions. In this study, we report on the uptake of mRNA by CD8α+ cross-presenting TiDCs upon its intratumoral (IT) delivery. We exploited this property to deliver mRNA encoding the co-stimulatory molecule CD70, the activation stimuli CD40 ligand, and constitutively active Toll-like receptor 4, referred to as TriMix mRNA. We show that TiDCs are reprogrammed to mature antigen-presenting cells that migrate to tumor-draining lymph nodes (TDLNs). TriMix stimulated antitumor T-cell responses to spontaneously engulfed cancer antigens, including a neoepitope. We showed in various mouse cancer models that IT delivery of TriMix mRNA results in systemic therapeutic antitumor immunity. Finally, we showed that the induction of antitumor responses critically depends on TiDCs, whereas it only partially depends on TDLNs. As such we provide a platform and a mechanistic rationale for the clinical testing of IT administration of TriMix mRNA., SCOPUS: ar.j, info:eu-repo/semantics/published

Background: Ipilimumab (Ipi) improves the survival of advanced melanoma patients with an incremental long-term benefit in 10-15 % of patients. A tumor signature that correlates with this survival benefit could help optimizing individualized treatment strategies. Methods: Freshly frozen melanoma metastases were collected from patients treated with either Ipi alone (n: 7) or Ipi combined with a dendritic cell vaccine (TriMixDC-MEL) (n: 11). Samples were profiled by immunohistochemistry (IHC), whole transcriptome (RNA-seq) and methyl-DNA sequencing (MBD-seq). Results: Patients were divided in two groups according to clinical evolution: durable benefit (DB; 5 patients) and no clinical benefit (NB; 13 patients). 20 metastases were profiled by IHC and 12 were profiled by RNA- and MBD-seq. 325 genes were identified as differentially expressed between DB and NB. Many of these genes reflected a humoral and cellular immune response. MBD-seq revealed differences between DB and NB patients in the methylation of genes linked to nervous system development and neuron differentiation. DB tumors were more infiltrated by CD8+ and PD-L1+ cells than NB tumors. B cells (CD20+) and macrophages (CD163+) co-localized with T cells. Focal loss of HLA class I and TAP-1 expression was observed in several NB samples. Conclusion: Combined analyses of melanoma metastases with IHC, gene expression and methylation profiling can potentially identify durable responders to Ipi-based immunotherapy., SCOPUS: ar.j, info:eu-repo/semantics/published

There is increasing evidence that the effect of chemotherapy on tumor rejection is not cell autonomous but relies on the immune system. Indeed, several reports have shown that human and murine tumors respond to chemotherapeutic agents more efficiently when the host immune system is intact. In particular, we have shown that cyclophosphamide treatment of DBA/2 mice bearing P815 mastocytoma induces rejection and long term protection in a CD4- and CD8-dependent manner. We used this tumor model, as it is poorly immunogenic, expresses tumor-associated P1A and tumor-specific P1E antigens, encoded by germline and mutated genes, respectively, and allows the identification of some tumor-specific CD8+ T cells.We have previously reported that tumor regression correlates with selective infiltration of CD8+ T cells specific for P1E/H-2Kd antigen in tumor bed upon cyclophosphamide treatment. Unexpectedly, the proportion of CD8+ T cells specific for the tumor-associated antigen P1A in the context of H-2Ld decreases concomitantly, indicating that cyclophosphamide alters the repertoire of CD8+ T cells recognizing tumor antigens. Using P1A KO mice, we found that preferential activation of CD8+ T cells to P1E is not solely due to thymic negative selection. The major role of “mutated” antigens in tumor resistance has been recently highlighted in humans and raises an interesting question about the immune mechanisms of tumor rejection. Additionally to its effect on the specific immune response, cyclophosphamide promotes tumor infiltration by effector memory (P1E/H-2Kd)+ CD8+ T cells which are characterized by higher expression of KLRG1 and Eomes. Our data point to a role of IL-15 and type 1 IFNs for their development, as increased levels of IL-15 and IRF7 were measured in tumor after cyclophosphamide. IFNAR1 blockade interferes with the tumor rejection in 50% of mice and decreases the (P1E/H-2Kd)+ CD8+ T cell infiltration induced by cyclophosphamide, suggesting a role of this cytokine in, Option Biologie moléculaire du Doctorat en Sciences, info:eu-repo/semantics/nonPublished

The severity and intensity of autoimmune disease in Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) patients and in scurfy mice emphasizes the critical role played by thymus-derived regulatory T cells (tTregs) in maintaining peripheral immune tolerance. However, although tTregs are critical to prevent lethal autoimmunity and excessive inflammatory responses, their suppressive mechanism remains elusive. Here we demonstrate that tTregs selectively inhibit CD27/CD70-dependent Th1 priming, while leaving the IL-12-dependent pathway unaffected. Immunized mice depleted of tTregs showed an increased response of IFN-γ-secreting CD4+ T cells that was strictly reliant on a functional CD27/CD70 pathway. In vitro studies revealed that tTregs downregulate CD70 from the plasma membrane of dendritic cells (DCs) in a CD27-dependent manner. CD70 downregulation required contact between Tregs and DCs and resulted in endocytosis of CD27 and CD70 into the DC. These findings reveal a novel mechanism by which tTregs can maintain tolerance or prevent excessive, proinflammatory Th1 responses, in press, SCOPUS: ar.j, FLWIN, info:eu-repo/semantics/published

Peripheral blood T cells transduced with a tumor-specific T-cell receptor (TCR) face problems of auto-reactivity and lack of efficacy caused by cross-pairing of exogenous and endogenous TCR chains, as well as short term in vivo survival due to activation and growth factor-induced differentiation. We here studied an alternative strategy for the efficient generation of naive CD8 + T cells with a single TCR. TCR-transduced human postnatal thymus-derived and adult mobilized blood-derived hematopoietic progenitor cells (HPCs) were differentiated to CD4 + CD8 + double-positive T cells using OP9-Delta-like 1 (OP9-DL1) cultures. Addition of the agonist peptide induced double positive cells to cross-present the peptide, leading, in the absence of co-stimulation, to cell cycle arrest and differentiation into mature CD8 + T cells. Comprehensive phenotypic, molecular and functional analysis revealed the generation of naive and resting CD8 + T cells through a process similar to thymic positive selection. These mature T cells show a near complete inhibition of endogenous TCRA and TCRB rearrangements and express high levels of the introduced multimer-reactive TCR. Upon activation, specific cytokine production and efficient killing of tumor cells were induced. Using this strategy, large numbers of high-avidity tumor-specific naive T cells can be generated from readily available HPCs without TCR chain cross-pairing. 2014 Macmillan Publishers Limited., SCOPUS: ar.j, info:eu-repo/semantics/published

Background Criteria for good candidate antigens for immunotherapy of acute myeloid leukemia are high expression on leukemic stem cells in the majority of patients with acute myeloid leukemia and low or no expression in vital tissues. It was shown in vaccination trials that Receptor for Hyaluronic Acid Mediated Motility (RHAMM/HMMR) generates cellular immune responses in patients with acute myeloid leukemia and that these responses correlate with clinical benefit. It is not clear however whether this response actually targets the leukemic stem cell, especially since it was reported that RHAMM is expressed maximally during the G2/M phase of the cell cycle. In addition, tumor specificity of RHAMM expression remains relatively unexplored. Design and Methods Blood, leukapheresis and bone marrow samples were collected from both acute myeloid leukemia patients and healthy controls. RHAMM expression was assessed at protein and mRNA levels on various sorted populations, either fresh or after manipulation. Results High levels of RHAMM were expressed by CD34+CD38+ and CD34- acute myeloid leukemia blasts. However, only baseline expression of RHAMM was measured in CD34+CD38- leukemic stem cells, and was not different from that in CD34+CD38- hematopoietic stem cells from healthy controls. RHAMM was significantly up-regulated in CD34+ cells from healthy donors during in vitro expansion and during in vivo engraftment. Finally, we demonstrated an explicit increase in the expression level of RHAMM after in vitro activation of T cells. Conclusions RHAMM does not fulfill the criteria of an ideal target antigen for immunotherapy of acute myeloid leukemia. RHAMM expression in leukemic stem cells does not differ significantly from the expression in hematopoietic stem cells from healthy controls. RHAMM expression in proliferating CD34+ cells of healthy donors and activated T cells further compromises RHAMM-specific T-cell-mediated immunotherapy. © 2012 Ferrata Storti Foundation., SCOPUS: ar.j, info:eu-repo/semantics/published

Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Lentiviral based constructs represent a recent development in the generation of transgenic animals. The ease of use, and the fact that the same backbone vectors can be used to down-modulate endogenous gene expression and to produce transgenic animals overexpressing a gene of interest, have fuelled growing interest in this technology. In this study, we have used a lentiviral delivery system to generate transgenic mice expressing altered levels (up or downregulated) of a gene of interest. Although this lentiviral-based approach led to high levels of transgenesis and germ line transmission, a wide variation in transgene expression was observed in most first and second generation mouse lines. In particular, despite the segregation of integrants into single-copy expressing mouse lines, transgene expression appeared to be the target of epigenetic regulatory mechanism, often causing the coexistence of high and low transgene expressing cells within a given tissue such as blood peripheral lymphocytes. The establishment and analysis of large number of mouse lines may therefore be required to select a stable transgenic line with pancellular expression of a gene of interest using this lentiviral-based approach., JOURNAL ARTICLE, SCOPUS: ar.j, info:eu-repo/semantics/published

Melanoma patients were injected with various vaccines containing a MAGE-A3 peptide presented by HLA-DP4. Anti-MAGE-A3.DP4 T cells were not detectable in the blood before vaccination, but their frequencies after vaccination ranged from 2 x 10(-6) to 2 x 10(-3) among the CD4(+) blood T lymphocytes of the patients. The CD4(+) blood T lymphocytes that stained ex vivo with HLA-DP4 tetramers folded with the MAGE-A3 peptide were selected by flow cytometry and amplified under clonal conditions. About 5% of the CD4(+) T-cell clones that recognized the MAGE-A3.DP4 antigen had a CD25(+) phenotype in the resting state. These CD25(+) clones had a high capacity to suppress the proliferation of another T-cell clone after peptide stimulation in vitro. Most of them had high FOXP3 expression in the resting state and an unmethylated FOXP3 intron 1. They produced active transforming growth factor-beta but none of cytokines IFN-gamma, interleukin-2 (IL-2), IL-4, IL-5, and IL-10. About 20% of CD25(-) clones had a significant but lower suppressive activity. Most of the CD25(-) clonal populations contained cells that expressed FOXP3 in the resting state, but FOXP3 demethylation was not observed. We conclude that MAGE-A3.DP4 vaccination can produce CD4(+) T cells that may exert regulatory T-cell function in vivo., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

A critical factor determining the effectiveness of currently used dendritic cell (DC)-based vaccines is the DC activation or maturation status. We have recently shown that the T-cell stimulatory capacity of DCs pulsed with tumor-antigen-derived peptides can be considerably increased by activating the DCs through electroporation with mRNA encoding CD40 ligand, CD70, and a constitutively active Toll-like receptor 4 (TriMix DCs). Here, we investigate whether TriMix DCs can be coelectroporated with whole tumor-antigen-encoding mRNA., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Overexpression of CD95 (Fas/Apo-1) ligand (CD95L) has been shown to induce T cell tolerance but also, neutrophilic inflammation and rejection of allogeneic tissue. We explored the capacity of dendritic cells (DCs) genetically engineered to overexpress CD95L to induce an antitumor response. We first found that DCs overexpressing CD95L, in addition to MHC class I-restricted OVA peptides (CD95L-OVA-DCs), induced increased antigen-specific CD8(+) T cell responses as compared with DCs overexpressing OVA peptides alone. The enhanced T cell responses were associated with improved regression of a tumor expressing OVA, allowing survival of all animals. When DCs overexpressing CD95L (CD95L-DCs) were injected with the tumor expressing OVA, in vivo tumor proliferation was strikingly inhibited. A strong cellular apoptosis and a massive neutrophilic infiltrate developed in this setting. Neutrophil depletion prevented tumor regression as well as enhanced IFN-gamma production induced by CD95L-OVA-DCs. Furthermore, the CD8(+) T cell response induced by the coadministration of tumor cells and CD95L-DCs led to rejection of a tumor implanted at a distance from the DC injection site. In summary, DCs expressing CD95L promote tumor rejection involving neutrophil-mediated innate immunity and CD8(+) T cell-dependent adaptative immune responses., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Dendritic cells (DCs) are professional APCs that have a unique capacity to initiate primary immune responses, including tolerogenic responses. We have genetically engineered bone marrow-derived DCs to express the immunosuppressive cytokine IL-10 and tested the ability of these cells to control experimental asthma. A single intratracheal injection of OVA-pulsed IL-10-transduced DCs (OVA-IL-10-DCs) to naive mice before OVA sensitization and challenge prevented all of the cardinal features of airway allergy, namely, eosinophilic airway inflammation, airway hyperreactivity, and production of mucus, Ag-specific Igs, and IL-4. OVA-IL-10-DCs also reversed established experimental asthma and had long-lasting and Ag-specific effects. We furthermore showed, by using IL-10-deficient mice, that host IL-10 is required for mediating the immunomodulatory effects of OVA-IL-10-DCs and demonstrated a significant increase in the percentage of OVA-specific CD4(+)CD25(+)Foxp3(+)IL-10(+) regulatory T cells in the mediastinal lymph nodes of OVA-IL-10-DC-injected mice. Finally, adoptive transfer of CD4(+) mediastinal lymph node T cells from mice injected with OVA-IL-10-DCs protected OVA-sensitized recipients from airway eosinophilia upon OVA provocation. Our study describes a promising strategy to induce long-lasting Ag-specific tolerance in airway allergy., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Maturation of dendritic cells (DC) is a critical step in the induction of T cell responses and depends on the activation of NF-kappaB transcription factors. Therefore, inhibition of NF-kappaB activation has been proposed as a strategy to maintain DC in an immature stage and to promote immune tolerance. Herein, we generated murine myeloid DC expressing a mutated IkappaBalpha acting as a superrepressor of the classical NF-kappaB pathway (s-rIkappaB DC) to investigate the consequences of NF-kappaB inhibition on the ability of DC to prime T cell responses. Upon in vitro LPS activation, maturation of s-rIkappaB DC was profoundly impaired as indicated by defective up-regulation of MHC class II and costimulatory molecules and reduced secretion of IL-12 p70 and TNF-alpha. In contrast, after injection, s-rIkappaB DC had the same capacity as control DC to migrate to draining lymph node and to induce Th1- and Th2-type cytokine production in a MHC class II-incompatible host mice. Likewise, s-rIkappaB DC pulsed with OVA were as efficient as control DC to induce Ag-specific T cell responses in vivo. Indeed, further in vitro experiments established that s-rIkappaB DC undergo efficient maturation upon prolonged contact with activated T cells via the alternative pathway of NF-kappaB activation triggered at least partly by lymphotoxin beta receptor ligation and involving processing of p100/RelB complexes., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Human papillomavirus type 16 is commonly implicated in cervical cancers. The viral genome encodes potential targets like the oncoprotein E7, expressed in transformed cells but thought to represent a poorly immunogenic antigen. We describe in this work a DNA-based vaccination protocol aimed at inducing an efficient anti-E7 immune response in vivo. Plasmids allowing the expression of the E7 protein in distinct cellular compartments were generated and assayed in an in vivo model of tumor growth. Our data demonstrate that mice vaccinated with a plasmid encoding for an E7 protein fused to a domain of the MHC class II-associated invariant chain (IiE7) were protected against tumor challenge. Mice immunized against an ubiquitinated form of E7 (Ub(Ala)E7) failed to control tumor growth. Protection induced by IiE7 was correlated with the development of CD8+ CTL and required the presence of CD4+ cells. In vitro studies confirmed that the IiE7 fusion protein was expressed at high levels in the endosomal compartment of transfected cells, while the natural and the ubiquitin-modified form of E7 were mainly nuclear. The present study suggests that an efficient anti-tumor response can be induced in vivo by DNA constructs encoding for E7 protein forms localizing at the endosomal compartment., Journal Article, Research Support, Non-U.S. Gov't, FLWIN, info:eu-repo/semantics/published

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SCOPUS: le.j, FLWIN, info:eu-repo/semantics/published

Aims/hypothesis. In the human pancreas, a close topographic relationship exists between duct cells and beta cells. This explains the high proportion of duct cells in isolated human islet preparations. We investigated whether human duct cells are a source of TNFα-mediated interactions with beta cells and immune cells. This cytokine has been implicated in the development of autoimmune diabetes in mice. Methods. Human duct cells were isolated from donor pancreases and examined for their ability to produce TNFα following a stress-signalling pathway. Duct-cell-released TNFα was tested for its in vitro effects on survival of human beta cells and on activation of human dendritic cells. Results. Exposure of human pancreatic duct cells to interleukin-1β (IL-1β) induces TNFα gene expression, synthesis of the 26,000 Mr TNFα precursor and conversion to the 17,000 Mr mature form, which is rapidly released. This effect is NO-independent and involves p38 MAPK and NF-κB signalling. Duct-cell-released TNFα contributed to cytokine-induced apoptosis of isolated human beta cells. It also induced activation of human dendritic cells. Conclusions/interpretation. Human pancreatic duct cells are a potential source of TNFα that can cause apoptosis of neighbouring beta cells and initiate an immune response through activation of dendritic cells. They may thus actively participate in inflammatory and immune processes that threaten beta cells during development of diabetes or after human islet cell grafts have been implanted., SCOPUS: ar.j, info:eu-repo/semantics/published

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-γ production by NK cells, correlating with an enhanced antitumor response. These data point to a novel mode of antitumor action for Gleevec., SCOPUS: ar.j, info:eu-repo/semantics/published

High-dose therapy (HDT) and autologous transplantation prolongs remission duration and survival in multiple myeloma (MM), but relapse still occurs at a median of 2 years post-HDT. In order to investigate whether the number of residual tumour cells in the bone marrow (BM) after transplantation can predict the duration of response, a quantitative allele-specific oligonucleotide polymerase chain reaction (qASO-PCR) assay was used to measure tumour load in BM at 3-6 months post-HDT in 67 patients. The method of maximally selected log-rank statistics was used to test for the existence of a cut-off value in the BM tumour load data set. A cut-off value with respect to progression-free survival (PFS) was identified (P = 0.001). The estimated threshold for placing patients into a 'good' or 'bad' prognostic group was 0.015% (n = 22 and 38 respectively) with a median PFS of 64 months vs. 16. Multivariate analysis showed grouping by PCR result to be an independent prognostic factor for PFS (estimated hazard ratio after shrinkage, 3.91). This study identifies for the first time a threshold of the post-HDT tumour load with prognostic significance for PFS in MM. Quantitative molecular assessment thus may help to identify those patients who are in need of further treatment early after autologous transplantation., SCOPUS: ar.j, FLWIN, info:eu-repo/semantics/published

There is increasing evidence that dendritic cells (DC) display opposite functions in the immune system, as they may induce immunity or tolerance depending on intrinsic and environmental factors. In mice, adoptive transfer of mature DC pulsed extracorporeally with antigen induces the development of antigen-specific Th1- and Th2-type CD4(+) cells. In this work, we compared the adjuvant properties of immature (freshly isolated) and mature (cultured) splenic DC in vivo. Our data show that injection of either cell population induces the clonal expansion of CD4(+) T cells but that only mature DC trigger their differentiation into effector cells producing IFN-gamma. In contrast, transfer of immature DC provokes the development of intermediates in the differentiation process, similar to the central memory cells. These observations, together with data in the literature, suggest that DC may induce tolerance, memory, or immunity depending on their maturation state., Journal Article, Research Support, Non-U.S. Gov't, FLWIN, info:eu-repo/semantics/published

Dendritic cells (DCs) genetically engineered to overexpress CD95 (Fas) ligand (CD95L-DC) were proposed as tools to induce peripheral tolerance to alloantigens. Herein, we observed that CD95L-DC obtained after retroviral gene transfer in bone marrow (BM) precursors derived from CD95-deficient (lpr/lpr) mice elicit much stronger allospecific type 1 helper T-cell and cytotoxic T-cell activities than control DCs upon injection in vivo, although they induce lower T-cell responses in vitro. Indeed, a single injection of CD95L-DC prepared from C57BL/6 mice was sufficient to prime bm13 recipients for acute rejection of C57BL/6 skin allografts that were otherwise tolerated in the context of this single weak major histocompatibility complex (MHC) class I incompatibility. Massive neutrophil infiltrates depending on interleukin (IL)-1 signaling were observed at sites of CD95L-DC injection. Experiments in IL-1 receptor-deficient mice or in animals injected with depleting anti-Gr1 monoclonal antibody (mAb) established that neutrophil recruitment is required for the development of vigorous T-cell responses after injection of CD95L-DC in vivo., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Dendritic cells (DC) are the most potent presenters of alloantigens and therefore are responsible for the induction of allograft rejection. Genetic modifications of DC allowing the expression of a tolerogenic molecule may render them immunosuppressive. We transduced bone marrow-derived DC with recombinant MFG retrovirus encoding either viral interleukin (vIL)-10 or Fas ligand (FasL) to induce transplantation tolerance. Up to 10 ng/ml of bioactive vIL-10 was produced by DC after transfer of the corresponding gene. Although the inhibitory properties of vIL-10-transduced DC were revealed in vitro in a mixed lymphocyte culture, no clear down-regulation of the allogeneic response was observed in vivo after single or multiple injections of those DC overexpressing vIL-10. When we transduced wild-type bone marrow-derived DC with recombinant MFG retrovirus encoding murine FasL, cells quickly died, probably because of suicidal or fratricidal Fas-dependent death. Indeed, only DC from Fas-deficient lpr mice survived to FasL gene transfer. Those FasL-transduced lpr DC exhibited a strong cytotoxic activity against Fas-positive targets in vitro. DC overexpressing FasL did not behave as immunosuppressive DC in vivo. The subcutaneous injection of FasL+ lpr DC in MHC class II-disparate mice hyperactivated the allospecific proliferation of T cells in the draining lymph nodes compared with mice treated with control-transduced DC. These results argue against the development of FasL+ DC or vIL-10-secreting DC as immunosuppressive tools in vivo. The alternative pathways of T-cell activation triggered by these genetically modified DC need to be investigated., Journal Article, Review, info:eu-repo/semantics/published

It was observed that interferon beta (IFN-beta) prevents the down-regulation of the interleukin-3 receptor alpha chain (IL-3Ralpha), which spontaneously occurs during culture of human monocytes. The functionality of IL-3R was demonstrated by the fact that IL-3 rescued IFN-beta-treated monocytes from apoptosis. Monocytes cultured in the presence of IFN-beta and IL-3 acquire a dendritic morphology and express high levels of HLA antigen class I and class II and costimulatory molecules. When stimulated by either lipopolysaccharide or fibroblasts expressing CD40 ligand (CD40L) transfectants, dendritic cells (DCs) generated in IFN-beta and IL-3 secreted high levels of IL-6, IL-8, and tumor necrosis factor-alpha but low levels of IL-12 in comparison with DCs generated in IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF). In mixed leukocyte culture, IL-3-IFN-beta DCs induced a vigorous proliferative response of allogeneic cord blood T cells and elicited the production of high levels of IFN-gamma and IL-5 by naive adult CD4+ T cells. Finally, IL-3-IFN-beta DCs were found to produce much higher levels of IFN-alpha than IL-4-GM-CSF DCs in response to Poly (I:C) but not to influenza virus. It was concluded that monocytes cultured in the presence of IL-3 and IFN-beta differentiate into DCs with potent helper T-cell stimulatory capacity despite their low secretion of IL-12., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Autoimmune lymphoproliferative syndrome (ALPS) is a paediatric disease characterized by lymphoproliferation and autoimmunity. Most patients are known to carry heterozygous mutations of the TNFRSF6 gene leading to diminished Fas-mediated apoptosis and failure of activated lymphocytes to undergo apoptosis. A subgroup of patients without the TNFRSF6 gene mutation has similar defective apoptosis and clinical features. No effective treatment has been reported so far. Glucocorticoids, intravenous immunoglobulin and/or immunosuppressive drugs have usually led to only transient clinical improvement. Seven ALPS patients (two type Ia and five type III) were treated with the antimalarial drug Fansidar. No toxicity was observed. An objective response was seen in six of them and, in two, the treatment was stopped without reappearance of the symptoms. Moreover, a marked decrease in interleukin-10 levels was observed in two patients during the treatment. We found that the drug induced apoptosis in activated lymphocytes through activation of the mitochondrial apoptotic pathway., Journal Article, SCOPUS: ar.j, FLWIN, info:eu-repo/semantics/published

Increasing evidence indicates that dendritic cells (DCs) are the antigen-presenting cells of the primary immune response. However, several reports suggest that B lymphocytes could be required for optimal T cell sensitization. We compared the immune responses of wild-type and B cell-deficient (muMT) mice, induced by antigen emulsified in adjuvant or pulsed on splenic dendritic cells. Our data show that lymph node cells from both control and muMT animals were primed, but each released distinct cytokine profiles. Lymph node T cells from control animals secreted interferon (IFN)-gamma, interleukin (IL)-2, and IL-4, whereas those from muMT mice produced IFN-gamma and IL-2 but no IL-4. To test whether B cells may influence the T helper cell type 1 (Th1)/Th2 balance by affecting the function of DCs, we immunized mice by transferring antigen-pulsed DCs from wild-type or mutant mice. Injection of control DCs induced the secretion of IL-4, IFN-gamma, and IL-2, whereas administration of DCs from muMT animals failed to sensitize cells to produce IL-4. Analysis of IL-12 production revealed that DCs from muMT mice produce higher levels of IL-12p70 than do DCs from wild-type animals. These data suggest that B lymphocytes regulate the capacity of DCs to promote IL-4 secretion, possibly by downregulating their secretion of IL-12, thereby favoring the induction of a nonpolarized immune response., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

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The CD40 molecule expressed on endothelial cells has been shown to transduce activation signals resulting in upregulation of adhesion molecules. Herein, we studied the impact of CD40 engagement on the induction of tissue factor (TF)-dependent procoagulant activity (PCA) at the surface of human umbilical vein endothelial cells (HUVECs). First, we found that co-incubation of HUVECs with 3T6 fibroblasts transfected with the CD40L gene (3T6-CD40L) resulted in a clear induction of PCA which was not observed with control untransfected fibroblasts. The specificity of this finding was established by inhibition experiments using monoclonal antibodies (mAbs) blocking CD40 or CD40L. PCA induced by CD40 ligation was TF-related as it was not observed in factor VII-deficient plasma and was associated with the accumulation of TF mRNA. To investigate the role of CD40/CD40L interactions in the induction of endothelial cell PCA by lymphocytes, interferon (IFN)-gamma-stimulated EC were incubated with T cells in the absence or presence of anti-CD40 or anti-CD40L mAb. The 60-70% inhibition of PCA induced by these mAbs but not their isotype-matched control indicated that the CD40 pathway is involved in the induction of PCA resulting from interactions between activated HUVECs and T cells. We conclude that activation signals elicited by CD40 engagement on endothelial cells result in the induction of TF-dependent PCA. The CD40/CD40L pathway might therefore be involved in the development of prothrombic states during diseases associated with endothelial cell and T cell activation., Journal Article, SCOPUS: ar.j, info:eu-repo/semantics/published

Background and Methods: The implications of the detection of residual disease after treatment of acute lymphoblastic leukemia (ALL) are unclear. We conducted a prospective study at 11 centers to determine the predictive value of the presence or absence of detectable residual disease at several points in time during the first six months after complete remission of childhood ALL had been induced. Junctional sequences of T-cell-receptor or immunoglobulin gene rearrangements were used as clonal markers of leukemic cells. Residual disease was quantitated with a competitive polymerase-chain-reaction (PCR) assay. Of 246 patients enrolled at diagnosis and treated with a uniform chemotherapy protocol, 178 were monitored for residual disease with one clone-specific probe (in 74 percent) or more than one probe (in 26 percent). The median follow-up period was 38 months. Results: The presence or absence and level of residual leukemia were significantly correlated with the risk of early relapse at each of the times studied (P<0.001). PCR measurements identified patients at high risk for relapse after the completion of induction therapy (those with ≤10-2 residual blasts per 2x105 mononuclear bone marrow cells) or at later time points (those with ≤10-3 residual blasts). Multivariate analysis showed that as compared with immunophenotype, age, risk group (standard or very high risk), and white-cell count at diagnosis, the presence or absence and level of residual disease were the most powerful independent prognostic factors. Conclusions: Residual leukemia after induction of a remission is a powerful prognostic factor in childhood ALL. Detection of residual disease by PCR should be used to identify patients at risk for relapse and should be taken into account in considering alternative treatment., SCOPUS: ar.j, info:eu-repo/semantics/published

We evaluated the effects of interleukin (IL)-10 on the differentiation of dendritic cells (DC) obtained by culturing plastic-adherent peripheral blood mononuclear cells for 7 days in presence of granulocyte/macrophage-colony-stimulating factor (GM-CSF) + IL-4. The addition of IL-10 at the initiation of culture resulted in the generation of macrophage-like cells with expressing high levels of CD14 and decreased levels of CD1a and CD1c. Furthermore, cells generated in presence of IL-10 secreted lower levels of IL-12, but higher levels of IL-8 compared with DC generated in absence of IL-10, both spontaneously and after CD40 engagement. Finally, cells generated in presence of IL-10 were less efficient than DC in stimulating the production of IL-2, interferon-gamma, and IL-4 by allogeneic T cells. We conclude that IL-10 prevents the generation of DC induced by GM-CSF + IL-4 and favors the development of macrophages with a lower T cell stimulatory potential, but secreting higher levels of IL-8 than DC., Journal Article, Research Support, Non-U.S. Gov't, FLWIN, info:eu-repo/semantics/published

Several cytokines play crucial roles in Trypanosoma cruzi infection in mice, but the involvement of endogenous granulocyte-macrophage colony- stimulating factor (GM-CSF) is poorly documented. This report shows that T. cruzi infection of mice triggered an early and sharp increase in plasma GM- CSF during the ascending phase of parasitemia. The plasma GM-CSF concentration remained stable at the peak of parasitemia and subsequently increased in those mice that survived to the acute phase. GM-CSF level increased again sharply, while parasitemia was rapidly decreasing. Finally, GM-CSF was undetectable, soon after the disappearance of circulating parasites. Injection of T. cruzi-infected mice with neutralizing anti-GM- CSF monoclonal antibodies induced the early appearance of parasitemia and aggravated cumulative mortality. In contrast, recombinant mouse GM-CSF (rmGM-CSF) caused sharp decreases in both parasitemia and cumulative mortality in T. cruzi-infected mice. Peritoneal macrophages from rmGM-CSF- treated and infected or uninfected mice were less infected ex vivo than those from control mice. Taken together, these data demonstrate the protective action of endogenous GM-CSF in T. cruzi infection. Neutralization of endogenous GM-CSF aggravates infection, while exogenous rmGM-CSF decreases both parasitemia and host mortality., SCOPUS: ar.j, info:eu-repo/semantics/published

Interactions between membrane-bound molecules were previously shown to be involved in the induction of tissue factor-dependent monocyte procoagulant activity (PCA) by activated T cells. To investigate the potential role of the CD40/CD40 ligand (CD40L) pathway in this process, we first determined the effects of blocking anti-CD40 or anti-CD40L monoclonal antibodies (mAb) on the development of monocyte PCA during mixed lymphocyte reaction (MLR) between allogeneic peripheral blood mononuclear cells (PBMC). The strong inhibitory effect exerted by both mAb (mean percentages of inhibition: 88 and 91% for anti-CD40 and anti-CD40L mAb, respectively) indicates that CD40/CD40L interactions are required for the induction of PCA in MLR. These data led us to measure monocyte PCA after incubation of PBMC or purified monocytes with a stimulating anti-CD40 mAb (BL-C4) or with 3T6 fibroblasts transfected with the gene encoding CD40L. In both systems, we found that CD40 engagement strongly induced monocyte PCA which was related to tissue factor expression as shown by flow cytometric analysis. Finally, we observed that recombinant interleukin (IL)-10, which inhibits lipopolysaccharide-induced PCA, did not significantly influence CD40-dependent PCA. We conclude that CD40 engagement on monocytes induces tissue factor-dependent PCA through an IL-10-resistant pathway. These findings have implications for the control of coagulation events triggered by interactions between T cells and monocytes., Journal Article, Research Support, Non-U.S. Gov't, FLWIN, info:eu-repo/semantics/published

SCOPUS: cp.j, info:eu-repo/semantics/published

Staphylococcus enterotoxins bind class II MHC molecules on antigen presenting cells (APC) and stimulate T cells expressing appropriate V beta gene products. Although the role of non-TCR associated co-stimulatory receptors during antigen-specific T cell stimulation has been clearly established, the involvement of co-stimulatory activity in T cell activation by superantigens has been the matter of controversy. In this report, we examine the role of co-stimulation provided by selected APC populations in the response to bacterial exotoxins (staphylococcal enterotoxin A, staphylococcal enterotoxin B and toxic shock syndrome type 1). We demonstrate that the APC population able to activate naive T cells to IL-2 production is heterogeneous, comprising both adherent (presumably dendritic) and non-adherent (mostly B lymphocytes) cells. By stimulating naive T cells in the presence of graded doses of superantigens, we have observed that half-maximal IL-2 production was achieved at lower doses of superantigens in the presence of dendritic cells. Similarly, addition of antibodies to CD28 or B7.1-transfected cell lines increased the sensitivity of naive T cells to lower doses of superantigens. These observations indicate therefore that superantigens can be presented to naive T cells by APC displaying distinct levels of co-stimulatory activity, although with different efficacy. Thus, naive T cells are sensitive to CD28-mediated co-stimulation during superantigen-mediated responses but IL-2 production can be induced by high doses of superantigens in the presence of APC expressing weak co-stimulatory activity. These observations are compatible with the hypothesis that CD28-mediated signals participate in T cell activation by lowering T cell sensitivity to TCR ligands., SCOPUS: ar.j, info:eu-repo/semantics/published

The interaction between B7-1 and CD28 provides costimulatory signals not only for T cells but also for natural killer (NK) cells. Highly metastatic mouse T lymphoma cells (BW-Li) can escape from NK cell-mediated killing by expressing H-2D(k) molecules that negatively regulate NK lytic activity. We have analyzed whether B7-1:CD28 overrules the MHC class I-mediated inactivation of NK cells by transfecting BW-Li with the gene coding for B7- 1. Expression of B7-1 rendered BW-Li cells sensitive toward NK cells. The experimental metastatic capacity of the B7-1 transfectants was drastically reduced in both syngeneic AKR and SCID mice but could be restored in SCID-bg mice. These results provide direct evidence that B7-1 expression leads to NK- mediated elimination of metastasizing, NK-resistant tumor cells., SCOPUS: ar.j, info:eu-repo/semantics/published

SCOPUS: ar.j, FLWNA, info:eu-repo/semantics/published

We have previously found that an increased tumorigenicity and spontaneous metastatic potential of BW5147-derived T lymphoma cells was associated with a decrease in major histocompatibility complex (MHC) class I H-2Kk antigen expression. This suggested that H-2Kk antigens may control the tumorigenic potential of BW T lymphoma cells. Our current experiments aimed to prove this association by specifically altering H-2Kk expression by gene transfection. Transfected cells expressing a high level of H-2Kk antigens were significantly less tumorigenic and metastatic after subcutaneous inoculation. However, there was selection in vivo for cells expressing a reduced level of H-2Kk antigens, which concomitantly led to an increased tumorigenicity. These data further confirmed the strong association between H-2Kk expression and tumorigenicity. We subsequently tested whether the immune system is implicated in this phenomenon by inoculating the H-2Kk transfectants into irradiated, immunocompromised recipients. Our results indicate that the reduced tumorigenicity of the BW H-2Kk transfectants is due to an immune rejection mechanism, mediated by CD8+ immune effector cells, as revealed by in vivo depletion experiments with anti-CD8 antibodies. Hence, we hereby demonstrated that H-2Kk antigens increased the immunogenicity of BW cells, via a CD8-dependent mechanism, which consequently reduced their tumorigenicity. © 1994 Rapid Communications of Oxford Ltd., SCOPUS: ar.j, info:eu-repo/semantics/published

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There is a significant increase in the percentage and absolute number of activated lymphocytes (OKT10/+, IL2R1/+) after ECT treatment of major depressive disorder. There is an acute decrease in the absolute number of total lymphocytes, T8/+ and Leu11/+ cells one hour after a single ECT., SCOPUS: cp.j, FLWNA, info:eu-repo/semantics/published, Ontogenetic and Phylogenetic Mechanisms of Neuroimmunomodulation: From Molecular Biology to Psychosocial Sciences

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