Facultad de Ciencias
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- Dendritic cells adhere to and transmigrate across lymphatic endothelium in response to IFN-α(Wiley Vch Verlag Berlin, 2010) Dubrot, J. (Juan); Martinez-Forero, I. (Iván); Alfaro, C. (Carlos); Rouzaut, A. (Ana); Gonzalez, I. (Iranzu); Larrea, E. (Esther); Garasa, S. (Saray); Hervas-Stubbs, S. (Sandra); Melero, I. (Ignacio); Palazon, A. (Asís); Suarez, N. (Natalia); Teijeira, A. (Álvaro)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-α were used to model this phenomenon under inflammatory conditions. DC differentiated in the presence of either IFN-α2b or IFN-α5 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-α 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-α 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.
- Agonist anti-CD137 mAb act on tumor endothelial cells to enhance recruitment of activated T lymphocytes(American Association for Cancer Research, 2011) Dubrot, J. (Juan); Martinez-Forero, I. (Iván); Jure-Kunkel, M. (María); Perez-Gracia, J.L. (Jose Luis); Rouzaut, A. (Ana); Peñuelas-Sanchez, I. (Ivan); Morales-Kastresana, A. (Aizea); Luque, A. (Alfonso); Hervas-Stubbs, S. (Sandra); Ochoa, L. (Laura); Ochoa, M.C. (María Carmen); Dinchuk, J. (Joseph); Melero, I. (Ignacio); Palazon, A. (Asís); Martinez, A. (Alfredo); Roncal, C. (Carmen); Teijeira, A. (Álvaro)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.
- Lysine 63 polyubiquitination in immunotherapy and in cancer-promoting inflammation(American Asociation for Cancer Research, 2009) Dubrot, J. (Juan); Martinez-Forero, I. (Iván); Rouzaut, A. (Ana); Melero, I. (Ignacio); Palazon, A. (Asís)Covalent and reversible post-translational modifications of proteins are a common theme in signaling. Ubiquitin conjugation was originally described to target proteins to proteasomal degradation by ubiquitin polymerization involving lysine (K) 48 residues. Differently linked polymers of polyubiquitin have been found that modify proteins without targeting to proteasomal degradation. Instead this pathway creates docking sites for signaling scaffolds that are key to control the nuclear factor-kappaB (NF-kappaB) pathway. Indeed TRAF-2, TRAF-6, and TRAF-3 are E3 ubiquitin ligases that form K63-linked ubiquitin polymers. Therefore signaling via TNF family receptors, IL1R, IL-18R, T-cell receptor (TCR), and Toll-like receptors (TLR) use this type of post-translational modification. Specific enzymes exist (DUBs) that deactivate this system, degrading K63 polyubiquitin chains. Interestingly, mice deficient in these deubiquitinases develop autoimmunity and inflammation. In carcinogenesis, the K63 polyubiquitin pathway is possibly critical for inflammation-driven tumor promotion. The pathway is also critically involved in costimulation of tumor immunity/immunotherapy as well as in the biology of malignant cells themselves. The elements of this new signaling paradigm offer the opportunity for therapeutic exploitation and drug discovery.
- Direct effects of type I interferons on cells of the immune system(American Association for Cancer Research, 2011) Perez-Gracia, J.L. (Jose Luis); Rouzaut, A. (Ana); Bon, A. (Agnes) Le; Hervas-Stubbs, S. (Sandra); Melero, I. (Ignacio); Fernandez-Sanmamed, M. (Miguel)Type I interferons (IFN-I) are well-known inducers of tumor cell apoptosis and antiangiogenesis via signaling through a common receptor interferon alpha receptor (IFNAR). IFNAR induces the Janus activated kinase-signal transducer and activation of transcription (JAK-STAT) pathway in most cells, along with other biochemical pathways that may differentially operate, depending on the responding cell subset, and jointly control a large collection of genes. IFNs-I were found to systemically activate natural killer (NK) cell activity. Recently, mouse experiments have shown that IFNs-I directly activate other cells of the immune system, such as antigen-presenting dendritic cells (DC) and CD4 and CD8 T cells. Signaling through the IFNAR in T cells is critical for the acquisition of effector functions. Cross-talk between IFNAR and the pathways turned on by other surface lymphocyte receptors has been described. Importantly, IFNs-I also increase antigen presentation of the tumor cells to be recognized by T lymphocytes. These IFN-driven immunostimulatory pathways offer opportunities to devise combinatorial immunotherapy strategies.
- Delivery of immunostimulatory monoclonal antibodies by encapsulated hybridoma cells(Springer Verlag, 2010) Dubrot, J. (Juan); Hernandez, R.M. (Rosa María); Pedraz, J.L. (José Luis); Perez-Gracia, J.L. (Jose Luis); Rouzaut, A. (Ana); Hervas-Stubbs, S. (Sandra); Orive, G. (Gorka); Melero, I. (Ignacio); Palazon, A. (Asís); Portero, A. (Aitziber)Immunostimulatory monoclonal antibodies are immunoglobulins directed toward surface proteins of immune system cells that augment the immune response against cancer in a novel therapeutic fashion. Exogenous administration of the recombinant humanized immunoglobulins is being tested in clinical trials with agents of this kind directed at a variety of immune-controlling molecular targets. In this study, the encapsulation of antibody-producing hybridoma cells was tested in comparison with the systemic administration of monoclonal antibodies. Hybridomas producing anti-CD137 and anti-OX40 mAb were encapsulated in alginate to generate microcapsules containing viable cells that secrete antibody. Immobilized cells in vitro were able to release the rat immunoglobulin produced by the hybridomas into the supernatant. Microcapsules were implanted by injection into the subcutaneous tissue of mice and thereby provided a platform for viable secreting cells, which lasted for more than 1 week. The pharmacokinetic profile of the rat monoclonal antibodies following microcapsule implantation was similar to that attained following an intraperitoneal administration of the purified antibodies. The rat-mouse hybridoma cells did not engraft as tumors in immunocompetent mice, while they lethally xenografted in immunodeficient mice, if not microencapsulated. The antitumor therapeutic activity of the strategy was studied on established CT26 colon carcinomas resulting in complete tumor eradication in an elevated fraction of cases and strong tumor-specific CTL responses with either anti-CD137 or anti-OX40 producing hybridomas, thus offering proof of the concept. This form of administration permitted combinations of more than one immunostimulatory monoclonal antibody to exploit the synergistic effects such as those known to be displayed by anti-CD137 and anti-OX40 mAb.
- Intratumoral injection of interferon-α and systemic delivery of agonist anti-CD137 monoclonal antibodies synergize for immunotherapy(Wiley Blackwell, 2011) Berraondo, P. (Pedro); Dubrot, J. (Juan); Martinez-Forero, I. (Iván); Alfaro, C. (Carlos); Rouzaut, A. (Ana); Bon, A. (Agnes) Le; Azpilicueta, A. (Arantza); Hervas-Stubbs, S. (Sandra); Ochoa, M.C. (María Carmen); Melero, I. (Ignacio); Palazon, A. (Asís); Teijeira, A. (Álvaro)CD137 artificial costimulation results in complete tumor rejection in several mouse models. Type I interferons (IFN) exert antitumor effects through an array of molecular functions on malignant cells, tumor stroma and immune system cells. The fact that agonist anti-CD137 mAb induce tumor regressions in mice deficient in the unique receptor for Type I IFNs (IFNAR(-/-) ) indicated potential for treatment combinations. Indeed, combination of intratumor injections of mouse IFN-α and intraperitoneal injections of anti-CD137 mAb synergized as seen on subcutaneous lesions derived from the MC38 colon carcinoma, which is resistant to each treatment if given separately. Therapeutic activity was achieved both against lesions directly injected with IFN-α and against distant concomitant tumors. Experiments in bone marrow chimeras prepared with IFNAR(-/-) and WT mice concluded that expression of the receptor for Type I interferons is mainly required on cells of the hematopoietic compartment. Synergistic effects correlated with a remarkable cellular hyperplasia of the tumor draining lymph nodes (TDLNs). Enlarged TDLNs contained more plasmacytoid and conventional dendritic cells (DC) that more readily cross-presented. Importantly, numbers of both DC subtypes inversely correlated with the tumor size. Numbers of CD8 T cells specific for a dominant tumor antigen were increased at TDLNs by each separate treatment but only with slight augments due to the combination. Combined antitumor effects of the therapeutic strategy were also seen on subcutaneous TC-1 tumors established for 24 days before treatment onset. The described strategy is realistic because (i) agents of each kind are clinically available and (ii) equivalent procedures in humans are feasible.
- Carcinoma-derived interleukin-8 disorients dendritic cell migration without impairing T-cell stimulation(Public Library of Science, 2011) Dubrot, J. (Juan); Martinez-Forero, I. (Iván); Alfaro, C. (Carlos); Solano, S. (Sarai); Perez-Gracia, J.L. (Jose Luis); Rouzaut, A. (Ana); Bolaños, E. (Elixabet); Gonzalez-Hernandez, A. (Alvaro); Gurpide, A. (Alfonso); Hervas-Stubbs, S. (Sandra); Erro, L. (Lorena); Melero, I. (Ignacio); Palazon, A. (Asís); Suarez, N. (Natalia); Feijoo, E. (Esperanza)BACKGROUND: Interleukin-8 (IL-8, CXCL8) is readily produced by human malignant cells. Dendritic cells (DC) both produce IL-8 and express the IL-8 functional receptors CXCR1 and CXCR2. Most human colon carcinomas produce IL-8. IL-8 importance in malignancies has been ascribed to angiogenesis promotion. PRINCIPAL FINDINGS: IL-8 effects on human monocyte-derived DC biology were explored upon DC exposure to recombinant IL-8 and with the help of an IL-8 neutralizing mAb. In vivo experiments were performed in immunodeficient mice xenografted with IL-8-producing human colon carcinomas and comparatively with cell lines that do not produce IL-8. Allogenic T lymphocyte stimulation by DC was explored under the influence of IL-8. DC and neutrophil chemotaxis were measured by transwell-migration assays. Sera from tumor-xenografted mice contained increasing concentrations of IL-8 as the tumors progress. IL-8 production by carcinoma cells can be modulated by low doses of cyclophosphamide at the transcription level. If human DC are injected into HT29 or CaCo2 xenografted tumors, DC are retained intratumorally in an IL-8-dependent fashion. However, IL-8 did not modify the ability of DC to stimulate T cells. Interestingly, pre-exposure of DC to IL-8 desensitizes such cells for IL-8-mediated in vitro or in vivo chemoattraction. Thereby DC become disoriented to subsequently follow IL-8 chemotactic gradients towards malignant or inflamed tissue. CONCLUSIONS: IL-8 as produced by carcinoma cells changes DC migration cues, without directly interfering with DC-mediated T-cell stimulation.
- Secondary myelodysplastic syndrome after treatment for promyelocytic leukemia: clinical and genetic features of two cases(Elsevier, 2003) Patiño-García, A. (Ana); Rocha, E. (Eduardo); Panizo, C. (Carlos); Bendandi, M. (Maurizio); Lecumberri, R. (Ramón); Calasanz-Abinzano, M.J. (Maria Jose); Odero, M.D. (Maria Dolores)Acute promyelocytic leukemia (APL) represents a biologic and clinically well-defined subtype of acute nonlymphocytic leukemia with specific morphologic and karyotypic characteristics. Although secondary leukemia and myelodysplastic syndromes (MDS) are the most frequent secondary neoplasms following chemotherapy for acute leukemia, their development after complete remission in patients with APL is uncommon. We describe the clinical and genetic features of two APL patients who achieved CR after chemotherapy and all-trans retinoid acid treatment and subsequently developed a MDS. Therapy-related MDS karyotype changes such as abnormalities of chromosomes 5 and 7 were found in the cytogenetic analysis. Since TP53 alteration was detected in one case, possible implications of these findings in the onset of MDS are discussed.
- Lack of Bcr-Abl point mutations in chronic myeloid leukemia patients in chronic phase before imatinib treatment is not predictive of response(Ferrata Storti Foundation, 2003) Montiel-Duarte, C. (Cristina); Andreu, E.J. (Enrique José); Fernandez-Luna, J.L. (J.L.); Larrayoz, M.J. (María J.); Prosper-Cardoso, F. (Felipe); Calasanz-Abinzano, M.J. (Maria Jose); Odero, M.D. (Maria Dolores); Aguirre-Ena, X. (Xabier); Fontalba, A. (A.)
- Simultaneous translocations of FGFR3/MMSET and CCND1 into two different IGH alleles in multiple myeloma: lack of concurrent activation of both proto-oncogenes(Elsevier, 2007) Cigudosa, J.C. (Juan Cruz); Largo, C. (Cristina); Siebert, R. (Reiner); Lahortiga, I. (Idoya); Larrayoz, M.J. (María J.); Prosper-Cardoso, F. (Felipe); Calasanz-Abinzano, M.J. (Maria Jose); Odero, M.D. (Maria Dolores); Martin-Subero, J.I. (Jose Ignacio); Saez, B. (Borja)The simultaneous occurrence of two different translocations affecting both alleles of the IGH gene has rarely been reported in multiple myeloma. In such a case, two different oncogenes might become transcriptionally deregulated. To investigate this hypothesis, we have characterized the plasma cell leukemia cell line SK-MM2 and a primary myeloma both carrying simultaneous IGHeFGFR3/MMSET and IGHeCCND1 fusions as shown by multicolor fluorescence in situ hybridization. Remarkably, quantitative real-time polymerase chain reaction demonstrated that only one of the oncogene loci was transcriptionally upregulated in both instances. Moreover, the upregulated oncogenes differed between both samples. Thus, biallelic IGH translocations might exert different pathogenetic effects in plasma cell disorders.