Morales-Kastresana, A. (Aizea)
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- Cancer immunotherapy with immunomodulatory anti-CD137 and anti-PD-1 monoclonal antibodies requires Batf3-dependent dendritic cells(American Association for Cancer Research, 2016) Jure-Kunkel, M. (María); Sancho, D. (David); Sanchez-Paulete, A.R. (Alfonso R.); Rodriguez-Ruiz, M.E. (María Esperanza); Cueto, F.J. (Francisco J.); Quetglas, J.I. (José Ignacio); Morales-Kastresana, A. (Aizea); Aznar, M.A. (María Ángela); Azpilicueta, A. (Arantza); Melero, I. (Ignacio); Martinez-Lopez, M. (María); Labiano, S. (Sara)Weak and ineffective antitumor cytotoxic T lymphocyte (CTL) responses can be rescued by immunomodulatory mAbs targeting PD-1 or CD137. Using Batf3−/− mice, which are defective for cross-presentation of cell-associated antigens, we show that BATF3-dependent dendritic cells (DC) are essential for the response to therapy with anti-CD137 or anti–PD-1 mAbs. Batf3−/− mice failed to prime an endogenous CTL-mediated immune response toward tumor-associated antigens, including neoantigens. As a result, the immunomodulatory mAbs could not amplify any therapeutically functional immune response in these mice. Moreover, administration of systemic sFLT3L and local poly-ICLC enhanced DC-mediated cross-priming and synergized with anti–CD137- and anti–PD-1–mediated immunostimulation in tumor therapy against B16-ovalbumin–derived melanomas, whereas this function was lost in Batf3−/− mice. These experiments show that cross-priming of tumor antigens by FLT3L- and BATF3-dependent DCs is crucial to the efficacy of immunostimulatory mAbs and represents a very attractive point of intervention to enhance their clinical antitumor effects.
- Nivolumab and urelumab enhance antitumor activity of human T lymphocytes engrafted in Rag2-/-IL2Rγnull immunodeficient mice(AACR, 2015) Mazzolini, G. (Guillermo); Jure-Kunkel, M. (María); Alfaro, C. (Carlos); Azpilikueta, A. (Arantza); Perez-Gracia, J.L. (Jose Luis); Martin-Algarra, S. (Salvador); Rodriguez, I. (Inmaculada); Rodriguez-Ruiz, M.E. (María Esperanza); Hidalgo, M. (Manuel); Morales-Kastresana, A. (Aizea); Sarno, F. (Francesca); Oñate, C. (Carmen); Melero, I. (Ignacio); Labiano, S. (Sara); Schalper, K.A. (Kurt A.); Korman, A.J. (Alan J.); Fernandez-Sanmamed, M. (Miguel)A current pressing need in cancer immunology is the development of preclinical model systems that are immunocompetent for the study of human tumors. Here, we report the development of a humanized murine model that can be used to analyze the pharmacodynamics and antitumor properties of immunostimulatory monoclonal antibodies (mAb) in settings where the receptors targeted by the mAbs are expressed. Human lymphocytes transferred into immunodeficient mice underwent activation and redistribution to murine organs, where they exhibited cell-surface expression of hCD137 and hPD-1. Systemic lymphocyte infiltrations resulted in a lethal CD4(+) T cell-mediated disease (xenograft-versus-host disease), which was aggravated when murine subjects were administered clinical-grade anti-hCD137 (urelumab) and anti-hPD-1 (nivolumab). In mice engrafted with human colorectal HT-29 carcinoma cells and allogeneic human peripheral blood mononuclear cells (PBMC), or with a patient-derived gastric carcinoma and PBMCs from the same patient, we found that coadministration of urelumab and nivolumab was sufficient to significantly slow tumor growth. Correlated with this result were increased numbers of activated human T lymphocytes producing IFNγ and decreased numbers of human regulatory T lymphocytes in the tumor xenografts, possibly explaining the efficacy of the therapeutic regimen. Our results offer a proof of concept for the use of humanized mouse models for surrogate efficacy and histology investigations of immune checkpoint drugs and their combinations.
- Molecular pathways: hypoxia response in immune cells fighting or promoting cancer(American Association for Cancer Research, 2012) Morales-Kastresana, A. (Aizea); Aragones, J. (Julián); Melero, I. (Ignacio); Palazon, A. (Asís); Ortiz-de-Landazuri, M. (Manuel)Both malignant and stromal components in tumors are influenced by the physiologic conditions of the microenvironment. Hypoxia is a prominent feature of solid tumors as a result of defective vascularization and intense metabolic activity. The gene-expression control mechanisms that adapt tissues to hypoxia are exploited by tumors to promote angiogenesis and vasculogenesis. The functions of infiltrating immune cells (macrophages and lymphocytes) and other stromal components are also influenced by a limited O(2) supply. Hypoxia-inducible factors (HIF) are the main molecular transcriptional mediators in the hypoxia response. The degradation and activity of HIF-1α and HIF-2α are tightly controlled by the fine-tuned action of oxygen-sensing prolyl and asparaginyl hydroxylase enzymes. Recent evidence indicates that hypoxia can modulate the differentiation and function of T lymphocytes and myeloid cells, skewing their cytokine-production profiles and modifying the expression of costimulatory receptors. This conceivably includes tumor-infiltrating lymphocytes. Hypoxia not only directly affects tumor-infiltrating leukocytes but also exerts effects on tumor cells and vascular cells that indirectly cause selective chemokine-mediated recruitment of suppressive and proangiogenic T-cell subsets. This review focuses on changes induced by hypoxia in immune cells infiltrating solid malignancies. Such changes may either promote or fight cancer, and thus are important for immunotherapy.
- Treatment with anti-CD137 mAbs causes intense accumulations of liver T cells without selective antitumor immunotherapeutic effects in this organ(Springer Verlag, 2010) Dubrot, J. (Juan); Martinez-Forero, I. (Iván); Jure-Kunkel, M. (María); Alfaro, C. (Carlos); Perez-Gracia, J.L. (Jose Luis); Morales-Kastresana, A. (Aizea); Hervas-Stubbs, S. (Sandra); Ochoa, M.C. (María Carmen); Melero, I. (Ignacio); Palazon, A. (Asís); Prieto, J. (Jesús); Milheiro, F. (Francisca); Romero-Trevejo, J.L. (José Lorenzo); Chen, L. (Lieping)BACKGROUND/AIMS: Cancer therapy with agonist anti-CD137 mAbs has been shown to induce immune-mediated tumor rejections in mice, and equivalent agents of this kind are currently being tested in cancer patients. Previous reports indicated that CD137 stimulation induced polyclonal infiltrates of T lymphocytes in the liver. This study characterizes the liver infiltrates and the target dependency of the phenomena and addresses the question of whether tumors nested in the liver are a more favorable target for CD137-based immunotherapy. METHODS: Liver infiltrates were studied with conventional histology and multiple color flow cytometry of total liver leukocytes. CD137(-/-) mice, mice with a single rearrangement of the TCR (OT-1 mice) and Rag(-/-) mice were used to clarify molecular requirements. Mice implanted with MC38 colon carcinomas either subcutaneously or inside the liver were used for comparative studies under treatment with agonist anti-CD137 mAbs. RESULTS: CD137 treatment caused mononuclear inflammation in the portal spaces of the liver, which gave rise to moderate increases in transaminases without signs of cholestasis. Marked increases in the numbers of CD8+ T cells were observed, including CD8+ T lymphocytes co-expressing CD11c. Infiltrates were absent in CD137(-/-) mice and mitigated in mice harboring a single transgenic TCR on their CD8 T cells. Despite the tumor-independent accumulation of T cells in the liver, immunotherapeutic effects were not more prominent against tumors located in this organ. CONCLUSIONS: Target-dependent effects of CD137 stimulation lead to liver infiltration with T cells, but lymphocyte enrichment in this organ does not privilege this site for immunotherapeutic effects against transplanted tumors.
- 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.
- Synergistic effects of CTLA-4 blockade with tremelimumab and elimination of regulatory T lymphocytes in vitro and in vivo(Wiley-Blackwell, 2011) Dubrot, J. (Juan); Martinez-Forero, I. (Iván); Alfaro, C. (Carlos); Perez-Gracia, J.L. (Jose Luis); Martin-Algarra, S. (Salvador); Bolaños, E. (Elixabet); Morales-Kastresana, A. (Aizea); Sangro, B. (Bruno); Gonzalez-Hernandez, A. (Alvaro); Hervas-Stubbs, S. (Sandra); Erro, L. (Lorena); Melero, I. (Ignacio); Palazon, A. (Asís); Suarez, N. (Natalia); Lecanda, F. (Fernando)Anti-CTLA-4 monoclonal antibodies (mAb) that block the interaction of CTLA-4 with CD80 and CD86 such as tremelimumab and ipilimumab are currently being tested in the clinic for cancer treatment exploiting their properties to de-repress tumor-specific cellular immunity. Addition of the fully human anti-CTLA-4 (tremelimumab) to cultures of human T cells with allogenic dendritic cells (DCs) did not increase proliferation. Magnetic bead-mediated elimination of CD4(+) CD25(+) regulatory T cells (T(reg)) before setting up those alloreactive cultures also largely failed to increase primary proliferation. In contrast, predepletion of CD4(+) CD25(+) T(reg) and culture in the presence of tremelimumab synergistically resulted in increased proliferation and DC:T-cell aggregation. These effects were much more prominent in CD4 than in CD8 T cells. 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-2Rγ(-/-) 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.
- Innate functions of immunoglobulin M lessen liver gene transfer with helper-dependent adenovirus(2014) Dubrot, J. (Juan); Morales-Kastresana, A. (Aizea); Unzu, C. (Carmen); Serrano-Mendioroz, I. (Irantzu); Azpilicueta, A. (Arantza); Ochoa, M.C. (María Carmen); Melero, I. (Ignacio); Fontanellas-Romá, A. (Antonio); Sampedro, A. (Ana); Martinez-Anso, E. (Eduardo)The immune system poses obstacles to viral vectors, even in the first administration to preimmunized hosts. We have observed that the livers of B cell-deficient mice were more effectively transduced by a helper-dependent adenovirus serotype-5 (HDA) vector than those of WT mice. This effect was T-cell independent as shown in athymic mice. Passive transfer of the serum from adenovirus-naïve WT to Rag1KO mice resulted in a reduction in gene transfer that was traced to IgM purified from serum of adenovirus-naïve mice. To ascribe the gene transfer inhibition activity to either adenoviral antigen-specific or antigen-unspecific functions of IgM, we used a monoclonal IgM antibody of unrelated specificity. Both the polyclonal and the irrelevant monoclonal IgM inhibited gene transfer by the HDA vector to either cultured hepatocellular carcinoma cells or to the liver of mice in vivo. Adsorption of polyclonal or monoclonal IgMs to viral capsids was revealed by ELISAs on adenovirus-coated plates. These observations indicate the existence of an inborn IgM mechanism deployed against a prevalent virus to reduce early post-infection viremia. In conclusion, innate IgM binding to adenovirus serotype-5 capsids restrains gene-transfer and offers a mechanism to be targeted for optimization of vector dosage in gene therapy with HDA vectors.
- Dendritic cells take up and present antigens from viable and apoptotic polymorphonuclear leukocytes(Public Library of Science, 2011) Martinez-Forero, I. (Iván); Alfaro, C. (Carlos); Perez-Gracia, J.L. (Jose Luis); Rodriguez, I. (Inmaculada); Bolaños, E. (Elixabet); Morales-Kastresana, A. (Aizea); Gonzalez-Hernandez, A. (Alvaro); Oñate, C. (Carmen); Hervas-Stubbs, S. (Sandra); Melero, I. (Ignacio); Palazon, A. (Asís); Perez, G. (Guiomar); Suarez, N. (Natalia); Fernandez-Sanmamed, M. (Miguel)Dendritic cells (DC) are endowed with the ability to cross-present antigens from other cell types to cognate T cells. DC are poised to meet polymorphonuclear leukocytes (PMNs) as a result of being co-attracted by interleukin-8 (IL-8), for instance as produced by tumor cells or infected tissue. Human monocyte-derived and mouse bone marrow-derived DC can readily internalize viable or UV-irradiated PMNs. Such internalization was abrogated at 4°C and partly inhibited by anti-CD18 mAb. In mice, DC which had internalized PMNs containing electroporated ovalbumin (OVA) protein, were able to cross-present the antigen to CD8 (OT-1) and CD4 (OT-2) TCR-transgenic T cells. Moreover, in humans, tumor cell debris is internalized by PMNs and the tumor-cell material can be subsequently taken up from the immunomagnetically re-isolated PMNs by DC. Importantly, if human neutrophils had endocytosed bacteria, they were able to trigger the maturation program of the DC. 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.