Nuestros investigadores

Publicaciones científicas más recientes (desde 2010)

Autores:  Zamarbide, M.; et al.
Revista: JOURNAL OF NEUROINFLAMMATION
ISSN 1742-2094  Vol. 16  Nº 1  2019 
Background Inflammation is a critical process for the progression of neuronal death in neurodegenerative disorders. Microglia play a central role in neuroinflammation and may affect neuron vulnerability. Next generation sequencing has shown the molecular heterogeneity of microglial cells; however, the variability in their response to pathological inputs remains unknown. Methods To determine the effect of an inflammatory stimulus on microglial cells, lipopolysaccharide (LPS) was administered peripherally to mice and the inflammatory status of the cortex, hippocampus, midbrain, and striatum was assessed. Microglial activation and interaction with the immune system were analyzed in single cell suspensions obtained from the different brain regions by fluorescence-activated cell sorting, next generation RNA sequencing, real-time PCR, and immunohistochemical techniques. Antigen-presenting properties of microglia were evaluated by the ability of isolated cells to induce a clonal expansion of CD4(+) T cells purified from OT-II transgenic mice. Results Under steady-state conditions, the midbrain presented a high immune-alert state characterized by the presence of two unique microglial subpopulations, one expressing the major histocompatibility complex class II (MHC-II) and acting as antigen-presenting cells and another expressing the toll-like receptor 4 (TLR4), and by the presence of a higher proportion of infiltrating CD4(+) T cells. This state was not detected in the cortex, hippocampus, or striatum. Systemic LPS administration induced a general increase in classic pro-inflammatory cytokines, in co-inhibitory programmed death ligand 1 (PD-L1), and in cytotoxic T lymphocyte antigen 4 (CTLA-4) receptors, as well as a decrease in infiltrating effector T cells in all brain regions. Interestingly, a specific immune-suppressive response was observed in the midbrain which was characterized by the downregulation of MHC-II microglial expression, the upregulation of the anti-inflammatory cytokines IL10 and TGF beta, and the increase in infiltrating regulatory T cells. Conclusions These data show that the midbrain presents a high immune-alert state under steady-state conditions that elicits a specific immune-suppressive response when exposed to an inflammatory stimulus. This specific inflammatory tone and response may have an impact in neuronal viability.
Autores: Ballesteros-Briones, M. C. ; Martisová, Eva; et al.
Revista: MOLECULAR THERAPY
ISSN 1525-0016  Vol. 27  Nº 11  2019  págs. 1892 - 1905
Immune checkpoint blockade has shown anti-cancer efficacy, but requires systemic administration of monoclonal antibodies (mAbs), often leading to adverse effects. To avoid toxicity, mAbs could be expressed locally in tumors. We developed adeno-associated virus (AAV) and Semliki Forest virus (SFV) vectors expressing anti-programmed death ligand 1 (aPDL1) mAb. When injected intratumorally in MC38 tumors, both viral vectors led to similar local mAb expression at 24 h, diminishing quickly in SFV-aPDL1-treated tumors. However, SFV-aPDL1 induced >40% complete regressions and was superior to AAV-aPDL1, as well as to aPDL1 mAb given systemically or locally. SFV-aPDL1 induced abscopal effects and was also efficacious against B16-ovalbumin (OVA). The higher SFV-aPDL1 antitumor activity could be related to local upregulation of interferon-stimulated genes because of SFV RNA replication. This was confirmed by combining local SFV-LacZ administration and systemic aPDL1 mAb, which provided higher antitumor effects than each separated agent. SFVaPDL1 promoted tumor-specific CD8 T cells infiltration in both tumor models. In MC38, SFV-aPDL1 upregulated co-stimulatory markers (CD137/OX40) in tumor CD8 T cells, and its combination with anti-CD137 mAb showed more pronounced antitumor effects than each single agent. These results indicate that local transient expression of immunomodulatory mAbs using non-propagative RNA vectors inducing type I interferon (IFN-I) responses represents a potent and safe approach for cancer treatment.
Autores: David Carneros; Eva M. Santamaria; et al.
Revista: FASEB JOURNAL
ISSN 0892-6638  Vol. 33  Nº 6  2019  págs. 7578 - 7587
Autores: Pascual, M.; Mena-Varas, M. ; Robles, Eloy Francisco; et al.
Revista: BLOOD
ISSN 0006-4971  Vol. 133  Nº 22  2019  págs. 2401 - 2412
Refractory or relapsed diffuse large B-cell lymphoma (DLBCL) often associates with the activated B-cell-like (ABC) subtype and genetic alterations that drive constitutive NF-kappa B activation and impair B-cell terminal differentiation. Here, we show that DNA damage response by p53 is a central mechanism suppressing the pathogenic cooperation of IKK2ca-enforced canonical NF-kappa B and impaired differentiation resulting from Blimp1 loss in ABC-DLBCL lymphomagenesis. We provide evidences that the interplay between these genetic alterations and the tumor microenvironment select for additional molecular addictions that promote lymphoma progression, including aberrant coexpression of FOXP1 and the B-cell mutagenic enzyme activation-induced deaminase, and immune evasion through major histocompatibility complex class II downregulation, PD-L1 upregulation, and T-cell exhaustion. Consistently, PD-1 blockade cooperated with anti-CD20-mediated B-cell cytotoxicity, promoting extended T-cell reactivation and antitumor specificity that improved long-term overall survival in mice. Our data support a pathogenic cooperation among NF-kappa B-driven prosurvival, genetic instability, and immune evasion mechanisms in DLBCL and provide preclinical proof of concept for including PD-1/PD-L1 blockade in combinatorial immunotherapy for ABC-DLBCL.
Autores: Ballesteros-Briones, M. C.; Martisová, Eva; et al.
Revista: HUMAN GENE THERAPY
ISSN 1043-0342  Vol. 30  Nº 11  2019  págs. A16 - A16
Autores: Azpilicueta, Arantza; et al.
Revista: CANCER RESEARCH
ISSN 0008-5472  Vol. 79  Nº 13  2019 
Autores: Trujillo, D. C.; Santamaria, E. M.; et al.
Revista: HEPATOLOGY
ISSN 0270-9139  Vol. 70  2019  págs. 1101A - 1102A
Autores: Ballesteros-Briones, M. C.; Martisová, Eva; et al.
Revista: MOLECULAR THERAPY
ISSN 1525-0016  Vol. 27  Nº 4  2019  págs. 268 - 268
Autores: Aritz Lasarte-Cia; et al.
Revista: FRONTIERS IN IMMUNOLOGY
ISSN 1664-3224  Vol. 9  Nº JAN  2018  págs. Article number: 68
A complex network of interactions exists between the immune, the olfactory, and the central nervous system (CNS). Inhalation of different fragrances can affect immunological reactions in response to an antigen but also may have effects on the CNS and cognitive activity. We performed an exploratory study of the immunomodulatory ability of a series of compounds representing each of the 10 odor categories or clusters described previously. We evaluated the impact of each particular odor on the immune response after immunization with the model antigen ovalbumin in combination with the TLR3 agonist poly I:C. We found that some odors behave as immunostimulatory agents, whereas others might be considered as potential immunosuppressant odors. Interestingly, the immunomodulatory capacity was, in some cases, strain-specific. In particular, one of the fragrances, carvone, was found to be immunostimulatory in BALB/c mice and immunosuppressive in C57BL/6J mice, facilitating or impairing viral clearance, respectively, in a model of a viral infection with a recombinant adenovirus. Importantly, inhalation of the odor improved the memory capacity in BALB/c mice in a fear-conditioning test, while it impaired this same capacity in C57BL/6J mice. The improvement in memory capacity in BALB/c was associated with higher CD3+ T cell infiltration into the hippocampus and increased local expression of mRNA coding for IL-1ß, TNF-¿, and IL-6 cytokines. In contrast, the memory impairment in C57BL/6 was associated with a reduction in CD3 numbers and an increase in IFN-¿. These data suggest an association between the immunomodulatory capacity of smells and their impact on the cognitive functions of the animals. These results highlight the potential of studying odors as therapeutic agents for CNS-related diseases.
Autores: Llopiz, Diana Isabel; Ruiz, Marta; et al.
Revista: ONCOIMMUNOLOGY
ISSN 2162-4011  Vol. 7  Nº 4  2018  págs. Article: e1409321
Tumor infiltrating lymphocytes have been associated with a better prognostic and with higher response rates in patients treated with checkpoint inhibiting antibodies, suggesting that strategies promoting tumor inflammation may enhance the efficacy of these currently available therapies. Our aim was thus to develop a new vaccination platform based on cold-inducible RNA binding protein (CIRP), an endogenous TLR4 ligand generated during inflammatory processes, and characterize whether it was amenable to combination with checkpoint inhibitors. In vitro, CIRP induced dendritic cell activation, migration and enhanced presentation of CIRP-bound antigens to T-cells. Accordingly, antigen conjugation to CIRP conferred immunogenicity, dependent on immunostimulatory and antigen-targeting capacities of CIRP. When applied in a therapeutic setting, vaccination led to CD8-dependent tumor rejection in several tumor models. Moreover, immunogenicity of this vaccination platform was enhanced not only by combination with additional adjuvants, but also with antibodies blocking PD-1/PD-L1, CTLA-4 and IL-10, immunosuppressive molecules usually present in the tumor environment and also induced by the vaccine. Therefore, priming with a CIRP-based vaccine combined with immune checkpoint-inhibiting antibodies rejected established B16-OVA tumors. Finally, equivalent activation and T-cell stimulatory effects were observed when using CIRP in vitro with human cells, suggesting that CIRP-based vaccination strategies could be a valuable clinical tool to include in combinatorial immunotherapeutic strategies in cancer patients.
Autores: Gorraiz, Marta; Lasarte-Cía, A.; et al.
Revista: ONCOTARGET
ISSN 1949-2553  Vol. 8  Nº 42  2017  págs. 71709 - 71724
Although T regulatory cells (Treg) are essential for the prevention of autoimmune diseases, their immunoregulatory function restrains the induction of immune responses against cancer. Thus, development of inhibitors of FOXP3, a key transcription factor for the immunosuppressive activity of Treg, might give new therapeutic opportunities. In a previous work we identified a peptide (named P60) able to enter into the cells, bind to FOXP3, and impair Treg activity in vitro and in vivo. Here we show that P60 binds to the intermediate region of FOXP3 and inhibits its homodimerization as well as its interaction with the transcription factor AML1. Alanine-scanning of P60 revealed the relevance of each position on FOXP3 binding, homodimerization, association with AML1 and inhibition of Treg activity. Introduction of alanine at positions 2, 5 and 11 improved the activity of the original P60, whereas alanine mutations at positions 1, 7, 8, 9, 10 and 12 were detrimental. Multiple mutation experiments allowed us to identify peptides with higher FOXP3 binding affinity and stronger biological activity than the original P60. Head to tail macrocyclization of peptide P60-D2A-S5A improved Treg inhibition and enhanced anti-tumor activity of anti-PD1 antibodies in a model of hepatocellular carcinoma. Introduction of a D-aminoacid at position 2 augmented significantly microsomal stability while maintained FOXP3 binding capacity and Treg inhibition in vitro. In vivo, when combined with the cytotoxic T-cell epitope AH1, it induced protection against CT26 tumor implantation. This study provides important structure¿function relationships essential for further drug design to inhibit Treg cells in cancer.
Autores: Sandra Hervas-Stubbs; et al.
Revista: PLOS ONE
ISSN 1932-6203  Vol. 12  Nº 9  2017  págs. e0185169
LAG3 receptor belongs to a family of immune-checkpoints expressed in T lymphocytes and other cells of the immune system. It plays an important role as a rheostat of the immune response. Focus on this receptor as a potential therapeutic target in cancer immunotherapy has been underscored after the success of other immune-checkpoint blockade strategies in clinical trials. LAG3 showcases the interest in the field of autoimmunity as several studies show that LAG3-targeting antibodies can also be used for the treatment of autoimmune diseases. In this work we describe the identification of a high-affinity LAG3 aptamer by High Throughput Sequencing SELEX in combination with a study of potential conserved binding modes according to sequence conservation by using 2D-structure prediction and 3D-RNA modeling using Rosetta. The aptamer with the highest accumulation of these conserved sequence motifs displays the highest affinity to LAG3 recombinant soluble proteins and binds to LAG3-expressing lymphocytes. The aptamer described herein has the potential to be used as a therapeutic agent, as it enhances the threshold of T-cell activation. Nonetheless, in future applications, it could also be engineered for treatment of autoimmune diseases by target depletion of LAG3-effector T lymphocytes.
Autores: Llopiz, Diana Isabel; Infante, S.; et al.
Revista: ONCOTARGET
ISSN 1949-2553  Vol. 8  Nº 2  2017  págs. 2659 - 2671
Vaccination induces immunostimulatory signals that are often accompanied by regulatory mechanisms such as IL-10, which control T-cell activation and inhibit vaccine-dependent antitumor therapeutic effect. Here we characterized IL10-producing cells in different tumor models treated with therapeutic vaccines. Although several cell subsets produced IL-10 irrespective of treatment, an early vaccine-dependent induction of IL-10 was detected in dendritic cells (DC). IL-10 production defined a DC population characterized by a poorly mature phenotype, lower expression of T-cell stimulating molecules and upregulation of PD-L1. These IL-10(+) DC showed impaired in vitro T-cell stimulatory capacity, which was rescued by incubation with IL-10R and PD-L1-inhibiting antibodies. In vivo IL-10 blockade during vaccination decreased the proportion of IL-10(+) DC and improved their maturation, without modifying PD-L1 expression. Similarly, PD-L1 blockade did not affect IL-10 expression. Interestingly, vaccination combined with simultaneous blockade of IL-10 and PD-L1 induced stronger immune responses, resulting in a higher therapeutic efficacy in tumor-bearing mice. These results show that vaccine-induced immunoregulatory IL-10(+) DC impair priming of antitumor immunity, suggesting that therapeutic vaccination protocols may benefit from combined targeting of inhibitory molecules expressed by this DC subset.
Autores: Fernandez-Poma, S. M.; Salas, Diego; et al.
Revista: CANCER RESEARCH
ISSN 0008-5472  Vol. 77  Nº 13  2017  págs. 3672 - 3684
Recent studies have found that tumor-infiltrating lymphocytes (TIL) expressing PD-1 can recognize autologous tumor cells, suggesting that cells derived from PD-1(+) TILs can be used in adoptive T-cell therapy (ACT). However, no study thus far has evaluated the antitumor activity of PD-1-selected TILs in vivo. In two mouse models of solid tumors, we show that PD-1 allows identification and isolationof tumor-specific TILs without previous knowledge of their antigen specificities. Importantly, despite the high proportion of tumor-reactive T cells present in bulk CD8 TILs before expansion, only T-cell products derived fromsorted PD-1(+), but not from PD-1(-) or bulk CD8 TILs, specifically recognized tumor cells. The fold expansion of PD-1(+) CD8 TILs was 10 times lower than that of PD-1(-) cells, suggesting that outgrowth of PD-1(-) cells was the limiting factor in the tumor specificity of cells derived from bulk CD8 TILs. The highly differentiated state of PD-1(+) cells was likely the main cause hampering ex vivo expansion of this subset. Moreover, PD-1 precisely identified marrow-infiltrating, myeloma-specific T cells in a mouse model of multiple myeloma. In vivo, only cells expanded from PD-1(+) CD8 TILs contained tumor progression, and their efficacy was enhanced by PDL-1 blockade. Overall, our data provide a rationale for the use of PD-1-selected TILs in ACT. (C) 2017 AACR.
Autores: Díaz-Lagares, A.; Méndez-González, J.; Sandra Hervas-Stubbs; et al.
Revista: CLINICAL CANCER RESEARCH
ISSN 1078-0432  Vol. 22  Nº 13  2016  págs. 3361-3371
PURPOSE: Lung cancer remains as the leading cause of cancer-related death worldwide, mainly due to late diagnosis. Cytology is the gold-standard method for lung cancer diagnosis in minimally invasive respiratory samples, despite its low sensitivity. We aimed to identify epigenetic biomarkers with clinical utility for cancer diagnosis in minimally/noninvasive specimens to improve accuracy of current technologies. EXPERIMENTAL DESIGN: The identification of novel epigenetic biomarkers in stage I lung tumors was accomplished using an integrative genome-wide restrictive analysis of two different large public databases. DNA methylation levels for the selected biomarkers were validated by pyrosequencing in paraffin-embedded tissues and minimally invasive and noninvasive respiratory samples in independent cohorts. RESULTS: We identified nine cancer-specific hypermethylated genes in early-stage lung primary tumors. Four of these genes presented consistent CpG island hypermethylation compared with nonmalignant lung and were associated with transcriptional silencing. A diagnostic signature was built using multivariate logistic regression model based on the combination of four genes:BCAT1, CDO1, TRIM58, andZNF177 Clinical diagnostic value was also validated in multiple independent cohorts and yielded a remarkable diagnostic accuracy in all cohorts tested. Calibrated and cross-validated epigenetic model predicts with high accuracy the probability to detect cancer in minimally and noninvasive samples. We demonstrated that this epigenetic signature achieved higher diagnostic efficacy in bronchial fluids as compared with conventional cytology for lung cancer diagnosis. CONCLUSION: Minimally invasive epigenetic biomarkers have emerged as promising tools for cancer diagnosis. The herein obtained epigenetic model in combination with current diagnostic protocols may improve early diagnosis and outcome of lung cancer patients.
Autores: Sandra Hervas-Stubbs; Soldevilla, M. M. ; et al.
Revista: ONCOTARGET
ISSN 1949-2553  Vol. 7  Nº 4  2016  págs. 4522 - 4530
TIM3 belongs to a family of receptors that are involved in T-cell exhaustion and Treg functions. The development of new therapeutic agents to block this type of receptors is opening a new avenue in cancer immunotherapy. There are currently several clinical trials ongoing to combine different immune-checkpoint blockades to improve the outcome of cancer patients. Among these combinations we should underline PD1:PDL1 axis and TIM3 blockade, which have shown very promising results in preclinical settings. Most of these types of therapeutic agents are protein cell-derived products, which, although broadly used in clinical settings, are still subject to important limitations. In this work we identify by HT-SELEX TIM3 non-antigenic oligonucleotide aptamers (TIM3Apt) that bind with high affinity and specificity to the extracellular motives of TIM3 on the cell surface. The TIM3Apt1 in its monomeric form displays a potent antagonist capacity on TIM3-expressing lymphocytes, determining the increase of IFN-¿ secretion. In colon carcinoma tumor-bearing mice, the combinatorial treatment of TIM3Apt1 and PDL1-antibody blockade is synergistic with a remarkable antitumor effect. Immunotherapeutic aptamers could represent an attractive alternative to monoclonal antibodies, as they exhibit important advantages; namely, lower antigenicity, being chemically synthesized agents with a lower price of manufacture, providing higher malleability, and antidote availability
Autores: Latasa, C.; Echeverz, M.; García, B.; et al.
Revista: PLOS ONE
ISSN 1932-6203  Vol. 11  Nº 8  2016  págs. e0161216
Salmonellosis is one of the most important bacterial zoonotic diseases transmitted through the consumption of contaminated food, with chicken and pig related products being key reservoirs of infection. Although numerous studies on animal vaccination have been performed in order to reduce Salmonella prevalence, there is still a need for an ideal vaccine. Here, with the aim of constructing a novel live attenuated Salmonella vaccine candidate, we firstly analyzed the impact of the absence of cyclic-di-GMP (c-di-GMP) in Salmonella virulence. C-di-GMP is an intracellular second messenger that controls a wide range of bacterial processes, including biofilm formation and synthesis of virulence factors, and also modulates the host innate immune response. Our results showed that a Salmonella multiple mutant in the twelve genes encoding diguanylate cyclase proteins that, as a consequence, cannot synthesize c-di-GMP, presents a moderate attenuation in a systemic murine infection model. An additional mutation of the rpoS gene resulted in a synergic attenuating effect that led to a highly attenuated strain, referred to as ¿XIII, immunogenic enough to protect mice against a lethal oral challenge of a S. Typhimurium virulent strain. ¿XIII immunogenicity relied on activation of both antibody and cell mediated immune responses characterized by the production of opsonizing antibodies and the induction of significant levels of IFN-¿, TNF-¿, IL-2, IL-17 and IL-10. ¿XIII was unable to form a biofilm and did not survive under desiccation conditions, indicating that it could be easily eliminated from the environment. Moreover, ¿XIII shows DIVA features that allow differentiation of infected and vaccinated animals. Altogether, these results show ¿XIII as a safe and effective live DIVA vaccine.
Autores: Garaude, J. , (Autor de correspondencia); Acín-Pérez, R. ; Martínez-Cano, S. ; et al.
Revista: NATURE IMMUNOLOGY
ISSN 1529-2908  Vol. 17  Nº 9  2016  págs. 1037 - 1045
Macrophages tightly scale their core metabolism after being activated, but the precise regulation of the mitochondrial electron-transport chain (ETC) and its functional implications are currently unknown. Here we found that recognition of live bacteria by macrophages transiently decreased assembly of the ETC complex I (CI) and CI-containing super-complexes and switched the relative contributions of CI and CII to mitochondrial respiration. This was mediated by phagosomal NADPH oxidase and the reactive oxygen species (ROS)-dependent tyrosine kinase Fgr. It required Toll-like receptor signaling and the NLRP3 inflammasome, which were both connected to bacterial viability-specific immune responses. Inhibition of CII during infection with Escherichia coli normalized serum concentrations of interleukin 1ß (IL-1ß) and IL-10 to those in mice treated with dead bacteria and impaired control of bacteria. We have thus identified ETC adaptations as an early immunological-metabolic checkpoint that adjusts innate immune responses to bacterial infection.
Autores: Pastor, Fernando; Soldevilla, M. M. ; et al.
Revista: MOLECULAR THERAPY - NUCLEIC ACIDS
ISSN 2162-2531  Vol. 5  2016  págs. e376 - e376
María Obdulia Rabal Gracia and Fernando Pastor Rodríguez, contributed equally to this work. ABSTRACT: Complementing Systematic Evolution of Ligands by EXponential Enrichment (SELEX) technologies with in silico prediction of aptamer binders has attracted a lot of interest in the recent years. We propose a workflow involving 2D structure prediction, 3D RNA modeling using Rosetta and docking to the target protein with 3dRPC for: (i) prediction of the binding mode of our two previously reported potent (Kd < 50 nmol/l) murine TIM3 aptamers, and (ii) the prioritization of TIM3 aptamers that were enriched in the SELEX workflow. The procedure was first validated in five different study cases. As a novelty, cluster analysis of the docked poses was carried out and shown to be useful in reproducing the binding mode or at least in identifying the binding site and the experimental aptamer-protein interactions. For TIM3, our therapeutic target of interest, a plausible binding site and binding mode was identified that might explain the lack of cross-reactivity in murine over human TIM-3. Concerning the prioritization of the aptamers, the inclusion of the cluster analysis as an additional criterion following a rank-by-rank approach is discussed and compared with the performance of the docking scoring function alone for two validation cases and for the prospective assessment of the novel aptamers as TIM3 binders.
Autores: Melero, Ignacio Javier; Sandra Hervas-Stubbs; et al.
Revista: GENE THERAPY
ISSN 0969-7128  Vol. 22  Nº 11  2015  págs. 856 - 865
Helper-dependent adenoviral (HDA) vectors constitute excellent gene therapy tools for metabolic liver diseases. We have previously shown that an HDA vector encoding human porphobilinogen deaminase (PBGD) corrects acute intermittent porphyria mice. Now, six non-human primates were injected in the left hepatic lobe with the PBGD-encoding HDA vector to study levels and persistence of transgene expression. Intrahepatic administration of 5 × 10(12) viral particles¿kg(-1) (10(10) infective units¿kg(-1)) of HDA only resulted in transient (¿14 weeks) transgene expression in one out of three individuals. In contrast, a more prolonged 90-day immunosuppressive regimen (tacrolimus, mycophenolate, rituximab and steroids) extended meaningful transgene expression for over 76 weeks in two out of two cases. Transgene expression under immunosuppression (IS) reached maximum levels 6 weeks after HDA administration and gradually declined reaching a stable plateau within the therapeutic range for acute porphyria. The non-injected liver lobes also expressed the transgene because of vector circulation. IS controlled anticapsid T-cell responses and decreased the induction of neutralizing antibodies. Re-administration of HDA-hPBGD at week +78 achieved therapeutically meaningful transgene expression only in those animals receiving IS again at the time of this second vector exposure. Overall, immunity against adenoviral capsids poses serious hurdles for long-term HDA-mediated liver transduction, which can be partially circumvented by pharmacological IS.
Autores: Concepción, A. R. ; Salas, J. T. ; Sáez, Elena; et al.
Revista: ONCOTARGET
ISSN 1949-2553  Vol. 6  Nº 30  2015  págs. 28588 - 28606
Primary biliary cirrhosis (PBC) is a chronic cholestatic disease of unknown etiopathogenesis showing progressive autoimmune-mediated cholangitis. In PBC patients, the liver and lymphocytes exhibit diminished expression of AE2/SLC4A2, a Cl-/HCO3- anion exchanger involved in biliary bicarbonate secretion and intracellular pH regulation. Decreased AE2 expression may be pathogenic as Ae2a,b(-/-) mice reproduce hepatobiliary and immunological features resembling PBC. To understand the role of AE2 deficiency for autoimmunity predisposition we focused on the phenotypic changes of T cells that occur over the life-span of Ae2a,b(-/-) mice. At early ages (1-9 months), knockout mice had reduced numbers of intrahepatic T cells, which exhibited increased activation, programmed-cell-death (PD)-1 expression, and apoptosis. Moreover, young knockouts had upregulated PD-1 ligand (PD-L1) on bile-duct cells, and administration of neutralizing anti-PD-L1 antibodies prevented their intrahepatic T-cell deletion. Older (¿ 10 months) knockouts, however, showed intrahepatic accumulation of cytotoxic CD8(+) T cells with downregulated PD-1 and diminished apoptosis. In-vitro DNA demethylation with 5-aza-2'-deoxycytidine partially reverted PD-1 downregulation of intrahepatic CD8(+) T cells from aged knockouts. CONCLUSION: Early in life, AE2 deficiency results in intrahepatic T-cell activation and PD-1/PD-L1 mediated deletion. With aging, intrahepatic CD8+ T cells epigenetically suppress PD-1, and their consequential expansion and further activation favor autoimmune cholangitis.
Autores: Gorraiz, Marta; et al.
Revista: JOURNAL OF IMMUNOLOGY
ISSN 0022-1767  Vol. 195  Nº 7  2015  págs. 3180 - 3189
Regulatory T cell (Treg) activity is modulated by a cooperative complex between the transcription factor NFAT and FOXP3, a lineage specification factor for Tregs. FOXP3/NFAT interaction is required to repress expression of IL-2, upregulate expression of the Treg markers CTLA4 and CD25, and confer suppressor function to Tregs. However, FOXP3 is expressed transiently in conventional CD4+ T cells upon TCR stimulation and may lead to T cell hyporesponsiveness. We found that a short synthetic peptide able to inhibit FOXP3/NFAT interaction impaired suppressor activity of conventional Tregs in vitro. Specific inhibition of FOXP3/NFAT interaction with this inhibitory peptide revealed that FOXP3 downregulates NFAT-driven promoter activity of CD40L and IL-17. Inhibition of FOXP3/NFAT interaction upregulated CD40L expression on effector T cells and enhanced T cell proliferation and IL-2, IFN-¿, IL-6, or IL-17 production in response to TCR stimulation. The inhibitory peptide impaired effector T cell conversion into induced Tregs in the presence of TGF-ß. Moreover, in vivo peptide administration showed antitumor efficacy in mice bearing Hepa129 or TC1 tumor cells when combined with sorafenib or with an antitumor vaccine, respectively. Our results suggest that inhibition of NFAT/FOXP3 interaction might improve antitumor immunotherapies.
Autores: Sandra Hervas-Stubbs; González-Aseguinolaza, Gloria; et al.
Revista: LIVER INTERNATIONAL
ISSN 1478-3223  Vol. 35  Nº 4  2014  págs. 1274 - 1289
BACKGROUND & AIMS: Adenoviral (Ad) vectors are currently one of the most efficient tools for in vivo gene transfer to the liver. However, anti-Ad immune responses limit the safety and efficacy of these vectors. The initial inflammatory reaction is a concern in terms of toxicity, and it favours the development of cellular and humoral responses leading to short transgene persistence and inefficient vector re-administrations. Therefore, safe and simple ways to interfere with these processes are needed. Study ways to deplete specific immune cell populations and their impact on liver-directed gene transfer. METHODS: First-generation Ad vectors encoding reporter genes (luciferase or ß-galactosidase) were injected intravenously into Balb/c mice. Kupffer cells and splenic macrophages were depleted by intravenous administration of clodronate liposomes. B lymphocytes, CD4(+) , CD8(+) T lymphocytes or NK cells were depleted by intraperitoneal injection of anti-M plus anti-D, anti-CD4, anti-CD8 or anti-asialo-GM1 antibodies respectively. Long-term evolution of luciferase expression in the liver was monitored by bioluminescence imaging. RESULTS: The anti-CD4 monoclonal antibody impaired cellular and humoral immune responses, leading to efficient vector re-administration. Clodronate liposomes had no impact on humoral responses but caused a 100-1000 fold increase in liver transduction, stabilized transgene expression, reduced the concentration of inflammatory cytokines, and inhibited lymphocyte activation. CONCLUSIONS: Transient CD4(+) T-cell depletion using antibodies is a clinically feasible procedure that allows efficient Ad redosing. Systemic administration of clodronate liposomes may further increase the safety and efficacy of vectors.
Autores: Arias MA; Jimenez de Bagües, MP; Aguilo, N; et al.
Revista: CELL REPORTS
ISSN 2211-1247  Vol. 8  Nº 2  2014  págs. 420-429
During bacterial sepsis, proinflammatory cytokines contribute to multiorgan failure and death in a process regulated in part by cytolytic cell granzymes. When challenged with a sublethal dose of the identified mouse pathogen Brucella microti, wild-type (WT) and granzyme A (gzmA)(-/-) mice eliminate the organism from liver and spleen in 2 or 3 weeks, whereas the bacteria persist in mice lacking perforin or granzyme B as well as in mice depleted of Tc cells. In comparison, after a fatal challenge, only gzmA(-/-) mice exhibit increased survival, which correlated with reduced proinflammatory cytokines. Depletion of natural killer (NK) cells protects WT mice from sepsis without influencing bacterial clearance and the transfer of WT, but not gzmA(-/-) NK, cells into gzmA(-/-) recipients restores the susceptibility to sepsis. Therefore, infection-related pathology, but not bacterial clearance, appears to require gzmA, suggesting the protease may be a therapeutic target for the prevention of bacterial sepsis without affecting immune control of the pathogen.
Autores: Dadaglio G; Fayolle C; Zhang X; et al.
Revista: JOURNAL OF IMMUNOLOGY
ISSN 0022-1767  Vol. 193  Nº 4  2014  págs. 1787-1798
Deciphering the mechanisms that allow the induction of strong immune responses is crucial to developing efficient vaccines against infectious diseases and cancer. Based on the discovery that the adenylate cyclase from Bordetella pertussis binds to the CD11b/CD18 integrin, we developed a highly efficient detoxified adenylate cyclase-based vector (CyaA) capable of delivering a large variety of Ags to the APC. This vector allows the induction of protective and therapeutic immunity against viral and tumoral challenges as well as against transplanted tumors in the absence of any added adjuvant. Two therapeutic vaccine candidates against human papilloma viruses and melanoma have been developed recently, based on the CyaA vector, and are currently in clinical trials. We took advantage of one of these highly purified vaccines, produced under good manufacturing practice-like conditions, to decipher the mechanisms by which CyaA induces immune responses. In this study, we demonstrate that CyaA binds both human and mouse CD11b(+) dendritic cells (DCs) and induces their maturation, as shown by the upregulation of costimulatory and MHC molecules and the production of proinflammatory cytokines. Importantly, we show that DCs sense CyaA through the TLR4/Toll/IL-1R domain-containing adapter-inducing IFN-ß pathway, independent of the presence of LPS. These findings show that CyaA possesses the intrinsic ability to not only target DCs but also to activate them, leading to the induction of strong immune responses. Overall, this study demonstrates that Ag delivery to CD11b(+) DCs in association with TLR4/Toll/IL-1R domain-containing adapter-inducing IFN-ß activation is an efficient strategy to promote strong specific CD8(+) T cell responses.
Autores: Sáez, Elena; et al.
Revista: EUROPEAN JOURNAL OF IMMUNOLOGY
ISSN 0014-2980  Vol. 44  Nº 5  2014  págs. 1341 - 1351
Mitogenic stimulation of lymphocytes involves alkalinization of intracellular pH (pHi ). Subsequent pHi regulation may involve HCO3 (-) extrusion through Cl(-) /HCO3 (-) exchangers and/or Na(+) -HCO3 (-) co-transporters with acid-loading capability. Abnormalities in these mechanisms could result in immune dysfunctions, as suggested by the CD8(+) T-cell expansion encountered in mice lacking Ae2 (a widely expressed acid loader with electroneutral and Na(+) -independent Cl(-) /HCO3 (-) anion-exchange activity). Here we report that CD8(+) T cells but not CD4(+) T cells or other lymphocyte populations, are crucially dependent on Ae2 for pHi regulation. While total lymphocytes (including isolated CD4(+) T cells) exhibit Ae1 expression and Na(+) -HCO3 (-) co-transport with acidifying potential, CD8(+) T cells lack these acid-loading mechanisms. In Ae2-KO mice, CD4(+) but not CD8(+) T cells upregulate these potential Ae2 surrogates. As a consequence, Ae2-KO CD8(+) T cells exhibit alkalinized pHi , and dramatically increase their pHi upon CD3 stimulation. Moreover, stimulated Ae2-deficient CD8(+) T cells show enhanced intracellular production of IL-2 and membrane expression of its receptor IL-2R¿, together with increased cell proliferation and activation. These findings demonstrate that CD8(+) T cells are critically dependent on Ae2 for pHi homeostasis and tuning of cell proliferation and activation. Ae2 thus constitutes a novel target to modulate CD8(+) T-cell responses.
Autores: Sandra Hervas-Stubbs; Riezu-Boj, José Ignacio; Mancheño, U.; et al.
Revista: JOURNAL OF IMMUNOLOGY
ISSN 0022-1767  Vol. 193  Nº 3  2014  págs. 1151 - 1161
Plasmacytoid dendritic cells (pDCs) are considered to be the principal type-I IFN (IFN-I) source in response to viruses, whereas the contribution of conventional DCs (cDCs) has been underestimated because, on a per-cell basis, they are not considered professional IFN-I-producing cells. We have investigated their respective roles in the IFN-I response required for CTL activation. Using a nonreplicative virus, baculovirus, we show that despite the high IFN-I-producing abilities of pDCs, in vivo cDCs but not pDCs are the pivotal IFN-I producers upon viral injection, as demonstrated by selective pDC or cDC depletion. The pathway involved in the virus-triggered IFN-I response is dependent on TLR9/MyD88 in pDCs and on stimulator of IFN genes (STING) in cDCs. Importantly, STING is the key molecule for the systemic baculovirus-induced IFN-I response required for CTL priming. The supremacy of cDCs over pDCs in fostering the IFN-I response required for CTL activation was also verified in the lymphocytic choriomeningitis virus model, in which IFN-ß promoter stimulator 1 plays the role of STING. However, when the TLR-independent virus-triggered IFN-I production is impaired, the pDC-induced IFNs-I have a primary impact on CTL activation, as shown by the detrimental effect of pDC depletion and IFN-I signaling blockade on the residual lymphocytic choriomeningitis virus-triggered CTL response detected in IFN-ß promoter stimulator 1(-/-) mice. Our findings reveal that cDCs play a major role in the TLR-independent virus-triggered IFN-I production required for CTL priming, whereas pDC-induced IFNs-I are dispensable but become relevant when the TLR-independent IFN-I response is impaired.
Autores: Melero, Ignacio Javier; Rodriguez-Madoz, Juan Roberto; et al.
Revista: CANCER RESEARCH
ISSN 0008-5472  Vol. 75  Nº 3  2014  págs. 497 - 507
Host responses are increasingly considered important for the efficacious response to experimental cancer therapies that employ viral vectors, but little is known about the specific nature of host responses required. In this study, we investigated the role of host type I interferons (IFN-I) in the efficacy of virally delivered therapeutic genes. Specifically, we used a Semliki Forest virus encoding IL12 (SFV-IL12) based on its promise as an RNA viral vector for cancer treatment. Intratumoral injection of SFV-IL12 induced production of IFN-I as detected in serum. IFN-I production was abolished in mice deficient for the IFN beta transcriptional regulator IPS-1 and partially attenuated in mice deficient for the IFN beta signaling protein TRIF. Use of bone marrow chimeric hosts established that both hematopoietic and stromal cells were involved in IFN-I production. Macrophages, plasmacytoid, and conventional dendritic cells were each implicated based on cell depletion experiments. Further, mice deficient in the IFN-I receptor (IFNAR) abolished the therapeutic activity of SFV-IL12, as did a specific antibody-mediated blockade of IFNAR signaling. Reduced efficacy was not caused by an impairment in IL12 expression, because IFNAR-deficient mice expressed the viral IL12 transgene even more strongly than wild-type (WT) hosts. Chimeric host analysis for the IFNAR involvement established a strict requirement in hematopoietic cells. Notably, although tumor-specific CD8 T lymphocytes expand
Autores: Sandra Hervas-Stubbs; Smerdou, Cristian;
Revista: ONCOIMMUNOLOGY
ISSN 2162-4011  Vol. 2  Nº 6  2013  págs. e24499
Do cancer patients responding to immunotherapy have immunological profiles that influence the therapeutic outcome, or do they develop efficient antitumor responses only upon immunotherapy? We came across this "chicken or the egg" dilemma when treating secondary liver tumors with Semliki Forest viruses expressing interleukin-12. In our system, the "egg," that is, the pre-treatment immunological profile, seemed to make the difference. The properties of an effective antitumor response were also defined.
Autores: Ochoa, María del Carmen; Mazzolini, G.; Sandra Hervas-Stubbs; et al.
Revista: CURRENT GENE THERAPY
ISSN 1566-5232  Vol. 13  Nº 1  2013  págs. 15 - 30
Interleukin-15 (IL-15) exerts powerful stimulatory effects on lymphocyte subsets that result in antiviral and antitumoral activities. The functions of this cytokine are mainly mediated in a cell-to-cell contact fashion termed IL-15 trans-presentation. This function is mediated by a cell which tethers IL-15 to its plasmatic membrane complexed to IL-15 receptor alpha (IL-15R¿). Such surface complexes interact with interleukin-2 receptor beta and gamma on the adjacent cell to elicit signaling. Unlike interleukin-2, IL-15 protects from activation-induced cell death and does not promote regulatory cells. These features underlie its activity against transplanted tumors and its adjuvanticity in tumor and viral vaccines. The GMP-manufactured recombinant protein is undergoing clinical trials but its rapid renal clearance calls for biotechnological strategies to increase molecular weight and ensure IL-15R¿. trans-presentation. Since early efforts with stable transfected tumor cells, IL-15 has been tested in a variety gene therapy approaches. Those mainly include transfer of expression cassettes to tumor cells, T cells, dendritic cells, vaccination sites and the liver as a biofactory organ. Detailed mechanistic knowledge of IL-15 biology is envisaged to make the most of a powerful immunotherapeutic tool ranked as one of the most promising for cancer immunotherapy.
Autores: Morales-Kastresana, A.; Catalán, E.; Sandra Hervas-Stubbs; et al.
Revista: JOURNAL FOR IMMUNOTHERAPY OF CANCER
ISSN 2051-1426  Vol. 1  2013  págs. 3
BACKGROUND: Treatment with agonist anti-CD137 (4-1BB) immunostimulatory monoclonal antibodies elicits complete tumor regressions in a number of transplanted hematological and solid malignancies in mice. Rejection is mainly dependent on cytotoxic T lymphocytes (CTL) and IFN¿, although a role for NK cells and dendritic cells has been observed in some tumor models. Rejection of EG7-derived thymomas has been shown to be CTL-dependent but not NK-dependent. FINDINGS: In this therapeutic setting, we show that both the perforin-granzyme and FasL effector systems are readily expressed by CD8(+) T lymphocytes infiltrating the EG7 lymphomas which are undergoing rejection. Using knock-out mice, we demonstrate that both effector cytolytic systems are involved in the execution of complete immune rejections against EG7 established tumors. In accordance, EG7 tumor cells were susceptible in vitro to both killing mechanisms acting in a synergistic fashion. CONCLUSIONS: CD137-elicited rejection of EG7-derived tumors involves the interplay of at least two final effector cytolytic mechanisms that act in cooperation.
Autores: Rodriguez-Madoz, Juan Roberto; Bezunartea, J.; et al.
Revista: JOURNAL OF IMMUNOLOGY
ISSN 0022-1767  Vol. 190  Nº 6  2013  págs. 2994 - 3004
Semliki Forest virus vectors expressing IL-12 (SFV-IL-12) were shown to induce potent antitumor responses against s.c. MC38 colon adenocarcinomas in immunocompetent mice. However, when MC38 tumors were implanted in liver, where colon tumors usually metastasize, SFV-IL-12 efficacy was significantly reduced. We reasoned that characterization of immune responses against intrahepatic tumors in responder and nonresponder animals could provide useful information for designing more potent antitumor strategies. Remarkably, SFV-IL-12 induced a high percentage of circulating tumor-specific CD8 T cells in all treated animals. Depletion studies showed that these cells were essential for SFV-IL-12 antitumor activity. However, in comparison with nonresponders, tumor-specific cells from responder mice acquired an effector-like phenotype significantly earlier, were recruited more efficiently to the liver, and, importantly, persisted for a longer period of time. All treated mice had high levels of functional specific CD8 T cells at 8 d posttreatment reflected by both in vivo killing and IFN-¿-production assays, but responder animals showed a more avid and persistent IFN-¿ response. Interestingly, differences in immune responses between responders and nonresponders seemed to correlate with the immune status of the animals before treatment and were not due to the treatment itself. Mice that rejected tumors were protected against tumor rechallenge, indicating that sustained memory responses are required for an efficacious therapy. Interestingly, tumor-specific CD8 T cells of responder animals showed upregulation of IL-15R¿ expression compared with nonresponders. These results suggest that SFV-IL-12 therapy could benefit from the use of strategies that could either upregulate IL-15R¿ expression or activate this receptor.
Autores: Rodríguez, I.; et al.
Revista: CLINICAL CANCER RESEARCH
ISSN 1078-0432  Vol. 19  Nº 22  2013  págs. 6151 - 6162
Purpose: Immunostimulatory monoclonal antibodies (ISmAb) that unleash antitumor immune responses are showing efficacy in cancer clinical trials. Anti-B7-H1 (PD-L1) monoclonal antibodies (mAb) block a critical inhibitory pathway in T cells, whereas anti-CD137 and OX40 mAbs provide T-cell costimulation. A combination of these ISmAbs (anti-CD137 + anti-OX40 + anti-B7-H1) was tested using a transgenic mouse model of multifocal and rapidly progressing hepatocellular carcinoma, in which c-myc drives transformation and cytosolic ovalbumin (OVA) is expressed in tumor cells as a model antigen. Experimental Design: Flow-cytometry and immunohistochemistry were used to quantify tumor-infiltrating lymphocytes (TIL) elicited by treatment and assess their activation status and cytolytic potential. Tolerance induction and its prevention/reversal by treatment with the combination of ISmAbs were revealed by in vivo killing assays. Results: The triple combination of ISmAbs extended survival of mice bearing hepatocellular carcinomas in a CD8-dependent fashion and synergized with adoptive T-cell therapy using activated OVA-specific TCR-transgenic OT-1 and OT-2 lymphocytes. Mice undergoing therapy showed clear increases in tumor infiltration by activated and blastic CD8(+) and CD4(+) T lymphocytes containing perforin/granzyme B and expressing the ISmAb-targeted receptors on their surface. The triple combination of ISmAbs did not result in enhanced OVA-specific cytotoxic T lymphocyte (CTL) activity but other antigens expressed by cell lines derived from such hepatocellular carcinomas were recognized by endogenous TILs. Adoptively transferred OVA-specific OT-1 lymphocytes into tumor-bearing mice were rendered tolerant, unless given the triple mAb therapy. Conclusion: Extension of survival and dense T-cell infiltrates emphasize the translational potential of combinational immunotherapy strategies for hepatocellular carcinoma.
Autores: Ochoa, María del Carmen; Rodriguez, I; Sandra Hervas-Stubbs; et al.
Revista: CANCER RESEARCH
ISSN 0008-5472  Vol. 73  Nº 1  2013  págs. 139-149
Interleukin (IL)-15 effects on CD8 T and natural killer (NK) lymphocytes hold promise to treat cancer. Fusion proteins have been engineered to provide IL-15 receptor alpha (IL-15R alpha) mediated trans-presentation to lymphocytes and extend the plasma half-life of the cytokine. In this study, we report on a triple fusion protein combining apolipoprotein A-I (Apo A-I), IL-15, and IL-15R alpha's sushi domain. Apo A-I conveys IL-15 to high-density lipoproteins (HDL), from which the cytokine is trans-presented by the IL-15R alpha's sushi domain. Such a construction was tested by hydrodynamic gene transfer to the liver of mice. Lethal toxicity was observed upon injection of 10 mu g of the expression plasmid. Mice died from an acute lymphocytic pneumonitis in which T and NK cells dominate a severe inflammatory infiltrate. Importantly, mice devoid of NK cells were not susceptible to such toxicity and mice lacking granzymes A and B also survived the otherwise lethal gene transfer. Lower plasmid doses (<2.5 mu g) were tolerated and dramatically increased the numbers of NK and memory CD8 T lymphocytes in the liver, spleen, and lungs, to the point of rescuing the deficiency of such lymphocyte subsets in IL-15R alpha(-/-) mice. Doses of plasmid within the therapeutic window successfully treated metastatic tumor models, including B16OVA lung metastasis of melanoma and MC38 colon cancer liver metastasis. Sushi-IL-15-Apo as a recombinant protein was also bioactive in vivo, became conjugated to HDL, and displayed immunotherapeutic effects against metastatic disease. Cancer Res; 73(1); 139-49. (C) 2012 AACR.
Autores: Sandra Hervas-Stubbs; et al.
Revista: HUMAN GENE THERAPY
ISSN 1043-0342  Vol. 23  Nº 12  2012  págs. 1258-1268
Replication-competent (oncolytic) adenoviruses (OAV) can be adapted as vectors for the delivery of therapeutic genes, with the aim of extending the antitumor effect beyond direct cytolysis. Transgene expression using these vectors is usually intense but short-lived, and repeated administrations are hampered by the rapid appearance of neutralizing antibodies (NAbs). We have studied the performance of monocytes as cell carriers to improve transgene expression in cancer models established in athymic mice and immunocompetent Syrian hamsters. Human and hamster monocytic cell lines (MonoMac6 and HM-1, respectively) were loaded with replication-competent adenovirus-expressing luciferase. Intravenous administration of these cells caused a modest increase in transgene expression in tumor xenografts, but this effect was virtually lost in hamsters. In contrast, intratumoral administration of HM-1 cells allowed repeated cycles of expression and achieved partial protection from NAbs in preimmunized hamsters bearing pancreatic tumors. To explore the therapeutic potential of this approach, HM-1 cells were loaded with a hypoxia-inducible OAV expressing the immunostimulatory cytokine interleukin-12 (IL-12). Three cycles of treatment achieved a significant antitumor effect in the hamster model, and transgene expression was detected following each administration, in contrast with the rapid neutralization of the free virus. We propose monocytes as carriers for multiple intratumoral administrations of armed OAVs.
Autores: Sandra Hervas-Stubbs; et al.
Revista: Journal of Translational Medicine
ISSN 1479-5876  Vol. 10  2012  págs.  222
These results indicate that our transient and intensive pharmacological immunosuppression fails to improve AAV5-based liver gene transfer in non-human primates. The reasons include an incomplete restraint of humoral immune responses to viral capsids that interfere with repeated gene transfer in addition to an intriguing MMF-dependent drug-mediated interference with liver transgene expression.
Autores: Sandra Hervas-Stubbs; Berasain, C; Golvano, J.; et al.
Revista: VACCINE
ISSN 0264-410X  Vol. 12  Nº 10  2012  págs. 867 - 871
This work shows that class II-linked humoral lack of response to an antigen can be overcome by joint immunization with the antigen and a T-helper cell determinant (TDh) well recognized by class II molecules of a non-responder individual. Thus, SJL/J mice (H-2s), which are non-responders to the S region of hepatitis B virus surface antigen (HBsAg), were rendered responders by joint immunization with a recombinant surface antigen, only composed of the S region, and a short synthetic TDh peptide well recognized by the H-2s restriction. By contrast, when this peptide is not recognized as TDh, as in B10M mice (H-2f restricted and also non-responders to the S region), no humoral response could be induced against the S region. These results have important implications for therapy and vaccination against hepatitis B virus as well as in enhancing the immunogenicity of other antigens.
Autores: Sandra Hervas-Stubbs; Mancheño, U; Riezu-Boj, José Ignacio; et al.
Revista: JOURNAL OF IMMUNOLOGY
ISSN 0022-1767  Vol. 189  Nº 7  2012  págs.  3299 - 3310
Autores: Ochoa, María del Carmen; Duitman, E. H.; et al.
Revista: PLOS ONE
ISSN 1932-6203  Vol. 7  Nº 12  2012 
Apolipoprotein A-I (Apo A-I) is a major component of high density lipoproteins (HDL) that transport cholesterol in circulation. We have constructed an expression plasmid encoding a chimeric molecule encompassing interleukin-15 (IL-15) and Apo A-I (pApo-hIL15) that was tested by hydrodynamic injections into mice and was co-administered with a plasmid encoding the sushi domain of IL-15R alpha (pSushi) in order to enhance IL-15 trans-presentation and thereby bioactivity. The pharmacokinetics of the Apo A-I chimeric protein were much longer than non-stabilized IL-15 and its bioactivity was enhanced in combination with IL-15R alpha Sushi. Importantly, the APO-IL-15 fusion protein was incorporated in part into circulating HDL. Liver gene transfer of these constructs increased NK and memory-phenotype CD8 lymphocyte numbers in peripheral blood, spleen and liver as a result of proliferation documented by CFSE dilution and BrdU incorporation. Moreover, the gene transfer procedure partly rescued the NK and memory T-cell deficiency observed in IL-15R alpha(-/-) mice. pApo-hIL15+ pSushi gene transfer to the liver showed a modest therapeutic activity against subcutaneously transplanted MC38 colon carcinoma tumors, that was more evident when tumors were set up as liver metastases. The improved pharmacokinetic profile and the strong biological activity of APO-IL-15 fusion protein holds promise for further development in combination with other immunotherapies.
Autores: Roncal, Carmen; calvayrac, o; et al.
Revista: Arteriosclerosis, thrombosis and vascular biology (Print)
ISSN 1079-5642  Vol. 32  Nº 6  2012  págs. 1477 - 1487
Autores: Bezunartea, Jaione; et al.
Revista: MOLECULAR THERAPY
ISSN 1525-0016  Vol. 20  Nº 9  2012  págs. 1664 - 1675
Intratumoral injection of Semliki Forest virus encoding interleukin-12 (SFV-IL-12) combines acute expression of IL-12 and stressful apoptosis of infected malignant cells. Agonist antibodies directed to costimulatory receptor CD137 (4-1BB) strongly amplify pre-existing cellular immune responses toward weak tumor antigens. In this study, we provide evidence for powerful synergistic effects of a combined strategy consisting of intratumoral injection of SFV-IL-12 and systemic delivery of agonist anti-CD137 monoclonal antibodies (mAbs), which was substantiated against poorly immunogenic B16 melanomas (B16-OVA and B16.F10) and TC-1 lung carcinomas. Effector CD8(beta)(+) T cells were sufficient to mediate complete tumor eradications. Accordingly, there was an intensely synergistic in vivo enhancement of cytotoxic T lymphocytes (CTL)-mediated immunity against the tumor antigens OVA and tyrosine-related protein-2 (TRP-2). This train of phenomena led to long-lasting tumor-specific immunity against rechallenge, attained transient control of the progression of concomitant tumor lesions that were not directly treated with SFV-IL-12 and caused autoimmune vitiligo. Importantly, we found that SFV-IL-12 intratumoral injection induces bright expression of CD137 on most tumor-infiltrating CD8(+) T lymphocytes, thereby providing more abundant targets for the action of the agonist antibody. This efficacious combinatorial immunotherapy strategy offers feasibility for clinical translation since anti-CD137 mAbs are already undergoing clinical trials and development of clinical-grade SFV-IL-12 vectors is in progress.
Autores: Alfaro, Carlos; et al.
Revista: Cancer Discovery
ISSN 2159-8274  Vol. 2  Nº 7  2012  págs. 608 - 623
Autores: Sandra Hervas-Stubbs; Pérez, José Luis; Rouzaut, Ana; et al.
Revista: CLINICAL CANCER RESEARCH
ISSN 1078-0432  Vol. 17  Nº 9  2011  págs. 2619 - 2627
Autores: Alfaro, Carlos; et al.
Revista: PLoS One
ISSN 1932-6203  Vol. 6  Nº 3  2011  págs. e17922
IL-8 as produced by carcinoma cells changes DC migration cues, without directly interfering with DC-mediated T-cell stimulation.
Autores: Alfaro, Carlos; et al.
Revista: PLoS One
ISSN 1932-6203  Vol. 6  Nº 12  2011  págs. e229300
Moreover, when mouse PMNs with E. coli in their interior are co-injected in the foot pad with DC, many DC loaded with fluorescent material from the PMNs reach draining lymph nodes. Using CT26 (H-2(d)) mouse tumor cells, it was observed that if tumor cells are intracellularly loaded with OVA protein and UV-irradiated, they become phagocytic prey of H-2(d) PMNs. If such PMNs, that cannot present antigens to OT-1 T cells, are immunomagnetically re-isolated and phagocytosed by H-2(b) DC, such DC productively cross-present OVA antigen determinants to OT-1 T cells. Cross-presentation to adoptively transferred OT-1 lymphocytes at draining lymph nodes also take place when OVA-loaded PMNs (H-2(d)) are coinjected in the footpad of mice with autologous DC (H-2(b)). In summary, our results indicate that antigens phagocytosed by short-lived PMNs can be in turn internalized and productively cross-presented by DC.
Autores: Ochoa, María del Carmen; Sandra Hervas-Stubbs; et al.
Revista: CANCER IMMUNOLOGY IMMUNOTHERAPY
ISSN 0340-7004  Vol. 60  Nº 5  2011  págs. 753 - 756
Autores: Suárez Fuentetaja, N.; Alfaro Alegría, C.; Dubrot Armendáriz, J.; et al.
Revista: International Journal of Cancer (Print)
ISSN 0020-7136  Vol. 129  Nº 2  2011  págs. 374 - 386
The synergy mechanism can be traced to enhanced CTLA-4 expression in effector cells as a result of T(reg) elimination, thereby offering more targets to the blocking antibody. Human T cells and allogenic DCs (derived both from healthy donors and advanced cancer patients) were coinjected in the peritoneum of Rag2(-/-) IL-2R¿(-/-) mice. In these conditions, tremelimumab injected intravenously did not significantly enhance alloreactive proliferation unless T(reg) cells had been predepleted. Synergistic effects in vivo were again largely restricted to the CD4 T-cell compartment. In addition, T(reg) depletion and CTLA-4 blockade synergistically enhanced specific cytotoxicity raised in culture against autologous EBV-transformed cell lines. Taken together, these experiments indicate that tremelimumab therapy may benefit from previous or concomitant T(reg) depletion
Autores: Alfaro, Carlos; Pérez, José Luis; et al.
Revista: The Journal of Immunology
ISSN 0022-1767  Vol. 187  Nº 11  2011  págs. 6130 - 6142
Twenty-four patients with metastatic cancer received two cycles of four daily immunizations with monocyte-derived dendritic cells (DC). DC were incubated with preheated autologous tumor lysate and subsequently with IFN-alpha, TNF-alpha, and polyinosinic:polycytidylic acid to attain type 1 maturation. One DC dose was delivered intranodally, under ultrasound control, and the rest intradermally in the opposite thigh. Cyclophosphamide (day -7), GM-CSF (days 1-4), and pegIFN alpha-2a (days 1 and 8) completed each treatment cycle. Pretreatment with cyclophosphamide decreased regulatory T cells to levels observed in healthy subjects both in terms of percentage and in absolute counts in peripheral blood. Treatment induced sustained elevations of IL-12 in serum that correlated with the output of IL-12p70 from cultured DC from each individual. NK activity in peripheral blood was increased and also correlated with the serum concentration of IL-12p70 in each patient. Circulating endothelial cells decreased in 17 of 18 patients, and circulating tumor cells markedly dropped in 6 of 19 cases. IFN-gamma-ELISPOT responses to DC plus tumor lysate were observed in 4 of 11 evaluated cases. Tracing DC migration with [(111)In] scintigraphy showed that intranodal injections reached deeper lymphatic chains in 61% of patients, whereas with intradermal injections a small fraction of injected DC was almost constantly shown to reach draining inguinal lymph nodes. Five patients experienced disease stabilization, but no objective responses were documented. This combinatorial immunotherapy strategy is safe and feasible, and its immunobiological effects suggest potential activity in patients with minimal residual disease. A randomized trial exploring this hypothesis is currently ongoing.
Autores: Alfaro, Carlos; Azpilicueta, Arantza; et al.
Revista: International Journal of Cancer (Print)
ISSN 0020-7136  Vol. 28  Nº 1  2011  págs. 105 - 118
Autores: Sandra Hervas-Stubbs; et al.
Revista: Cancer Research
ISSN 0008-5472  Vol. 71  Nº 3  2011  págs. 801 - 11
Agonist monoclonal antibodies (mAb) to the immune costimulatory molecule CD137, also known as 4-1BB, are presently in clinical trials for cancer treatment on the basis of their costimulatory effects on primed T cells and perhaps other cells of the immune system. Here we provide evidence that CD137 is selectively expressed on the surface of tumor endothelial cells. Hypoxia upregulated CD137 on murine endothelial cells. Treatment of tumor-bearing immunocompromised Rag(-/-) mice with agonist CD137 mAb did not elicit any measurable antiangiogenic effects. In contrast, agonist mAb stimulated tumor endothelial cells, increasing cell surface expression of the adhesion molecules intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin. When adoptively transferred into mice, activated T lymphocytes derived from CD137-deficient animals entered more avidly into tumor tissue after treatment with agonist mAb. This effect could be neutralized with anti-ICAM-1 and anti-VCAM-1 blocking antibodies. Thus, stimulation of CD137 not only enhanced T-cell activation but also augmented their trafficking into malignant tissue, through direct actions on the blood vessels that irrigate the tumor. Our findings identify an additional mechanism of action that can explain the immunotherapeutic effects of agonist CD137 antibodies
Autores: Rouzaut, Ana; Garasa, S.; et al.
Revista: European Journal of Immunology
ISSN 0014-2980  Vol. 40  Nº 11  2010  págs. 3054 - 3063
Migration of DC into lymphatic vessels ferries antigenic cargo and pro-inflammatory stimuli into the draining LN. Given that tissues under the influence of viral infections produce type I IFN, it is conceivable that these cytokines enhance DC migration in order to facilitate an antiviral immune response. Cultured lymphatic endothelium monolayers pretreated with TNF-alpha were used to model this phenomenon under inflammatory conditions. DC differentiated in the presence of either IFN-alpha 2b or IFN-alpha 5 showed enhanced adhesion to cultured lymphatic endothelial cells. These pro-adhesive effects were mediated by DC, not the lymphatic endothelium, and correlated with increased DC transmigration across lymphatic endothelial cell monolayers. Transmigration was guided by chemokines acting on DC, and blocking experiments with mAb indicated a role for LFA-1. Furthermore, incubation of DC with IFN-alpha led to the appearance of active conformation epitopes on the CD11a integrin chains expressed by DC. Differentiation of mouse DC in the presence of IFN-alpha also increased DC migration from inflammed footpads toward popliteal LN. Collectively, these results indicate a role for type I IFN in directing DC toward LN under inflammatory conditions.
Autores: Sandra Hervas-Stubbs; Riezu-Boj, José Ignacio; González, I; et al.
Revista: European Journal of Immunology
ISSN 0014-2980  Vol. 40  Nº 12  2010  págs. 3389 - 3402
Autores: Fontanellas, Antonio; Sandra Hervas-Stubbs; et al.
Revista: Molecular Therapy
ISSN 1525-0016  Vol. 18   Nº 4  2010  págs. 754 - 765
Autores: Ochoa, María del Carmen; Arina, A.; et al.
Revista: GENE THERAPY
ISSN 0969-7128  Vol. 17  Nº 5  2010  págs. 687 - 689
Autores: Milheiro, F; Alfaro, Carlos; et al.
Revista: CANCER IMMUNOLOGY IMMUNOTHERAPY
ISSN 0340-7004  Vol. 59  Nº 8  2010  págs. 1223 - 1233

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