Antitumor efficacy and reduced toxicity using an anti-CD137 Probody therapeutic.

Costimulation via CD137 (4-1BB) enhances antitumor immunity mediated by cytotoxic T lymphocytes. Anti-CD137 agonist antibodies elicit mild liver inflammation in mice, and the maximum tolerated dose of Urelumab, an anti-human CD137 agonist monoclonal antibody, in the clinic was defined by liver inflammation-related side effects. A protease-activated prodrug form of the anti-mouse CD137 agonist antibody 1D8 (1D8 Probody therapeutic, Pb-Tx) was constructed and found to be selectively activated in the tumor microenvironment. This construct, which encompasses a protease-cleavable linker holding in place a peptide that masks the antigen binding site, exerted antitumor effects comparable to the unmodified antibody but did not result in liver inflammation. Moreover, it efficaciously synergized with both PD-1 blockade and adoptive T-cell therapy. Surprisingly, minimal active Pb-Tx reached tumor-draining lymph nodes, and regional lymphadenectomy did not abrogate antitumor efficacy. By contrast, S1P receptor-dependent recirculation of T cells was absolutely required for efficacy. The preferential cleavage of the anti-CD137 Pb-Tx by tumor proteases offers multiple therapeutic opportunities, including neoadjuvant therapy, as shown by experiments in which the Pb-Tx is given prior to surgery to avoid spontaneous metastases.

Cytokines in clinical cancer immunotherapy.

Cytokines are soluble proteins that mediate cell-to-cell communication. Based on the discovery of the potent anti-tumour activities of several pro-inflammatory cytokines in animal models, clinical research led to the approval of recombinant interferon-alpha and interleukin-2 for the treatment of several malignancies, even if efficacy was only modest. These early milestones in immunotherapy have been followed by the recent addition to clinical practice of antibodies that inhibit immune checkpoints, as well as chimeric antigen receptor T cells. A renewed interest in the anti-tumour properties of cytokines has led to an exponential increase in the number of clinical trials that explore the safety and efficacy of cytokine-based drugs, not only as single agents, but also in combination with other immunomodulatory drugs. These second-generation drugs under clinical development include known molecules with novel mechanisms of action, new targets, and fusion proteins that increase half-life and target cytokine activity to the tumour microenvironment or to the desired effector immune cells. In addition, the detrimental activity of immunosuppressive cytokines can be blocked by antagonistic antibodies, small molecules, cytokine traps or siRNAs. In this review, we provide an overview of the novel trends in the cytokine immunotherapy field that are yielding therapeutic agents for clinical trials.

CD8 T cell priming in the presence of IFN-α renders CTLs with improved responsiveness to homeostatic cytokines and recall antigens: important traits for adoptive T cell therapy.

Previous mouse and human studies have demonstrated that direct IFN-α/ß signaling on naive CD8 T cells is critical to support their expansion and acquisition of effector functions. In this study, we show that human naive CD8 T cells primed in the presence of IFN-α possess a heightened ability to respond to homeostatic cytokines and to secondary Ag stimulation, but rather than differentiating to effector or memory CTLs, they preserve nature-like phenotypic features. These are qualities associated with greater efficacy in adoptive immunotherapy. In a mouse model of adoptive transfer, CD8 T cells primed in the presence of IFN-α are able to persist and to mediate a robust recall response even after a long period of naturally driven homeostatic maintenance. The long-lasting persistence of IFN-α-primed CD8 T cells is favored by their enhanced responsiveness to IL-15 and IL-7, as demonstrated in IL-15(-/-) and IL-7(-/-) recipient mice. In humans, exposure to IFN-α during in vitro priming of naive HLA-A2(+) CD8 T cells with autologous dendritic cells loaded with MART1(26-35) peptide renders CD8 T cells with an improved capacity to respond to homeostatic cytokines and to specifically lyse MART1-expressing melanoma cells. Furthermore, in a mouse model of melanoma, adoptive transfer of tumor-specific CD8 T cells primed ex vivo in the presence of IFN-α exhibits an improved ability to contain tumor progression. Therefore, exposure to IFN-α during priming of naive CD8 T cells imprints decisive information on the expanded cells that can be exploited to improve the efficacy of adoptive T cell therapy.

Low-dose ionizing γ-radiation elicits the extrusion of neutrophil extracellular traps.

PURPOSE: Cancer patients frequently undergo radiotherapy in their clinical management with unintended irradiation of blood vessels and copiously irrigated organs in which polymorphonuclear leukocytes circulate. Following the observation that such low doses of ionizing radiation are able to induce neutrophils to extrude neutrophil extracellular traps (NETs), we have investigated the mechanisms, consequences and the occurrence of such phenomena in patients undergoing radiotherapy. EXPERIMENTAL DESIGN: NETosis was analyzed in cultures of neutrophils isolated from healthy donors, cancer patients and cancer-bearing mice under confocal microscopy. Cocultures of radiation-induced NETs, immune effector lymphocytes and tumor cells were used to study the effects of irradiation-induced NETs on immune cytotoxicity. Radiation-induced NETs were intravenously injected to mice assessing their effects on metastasis. Circulating NETs in irradiated cancer patients were measured by ELISA methods detecting MPO-DNA complexes and citrullinated H3. RESULTS: Very low γ-radiation doses (0.5-1 Gy) given to neutrophils elicit NET formation in a manner dependent on oxidative stress, NADPH oxidase activity and autocrine interleukin-8. Radiation-induced NETs interfere with NK- and T-cell cytotoxicity. As a consequence, pre-injection of irradiation-induced NETs increases the number of successful metastases in mouse tumor models. Increases in circulating NETs were readily detected in two prospective series of patients following the first fraction of their radiotherapy courses. CONCLUSIONS: NETosis is induced by low-dose ionizing irradiation in a neutrophil-intrinsic fashion and radiation-induced NETs are able to interfere with immune-mediated cytotoxicity. Radiation-induced NETs foster metastasis in mouse models and can be detected in the circulation of patients undergoing conventional radiotherapy treatments.

The oncolytic adenovirus Delta-24-RGD in combination with ONC201 induces a potent antitumor response in pediatric high-grade and diffuse midline glioma models.

BACKGROUND: Pediatric high-grade gliomas (pHGGs), including diffuse midline gliomas (DMGs), are aggressive pediatric tumors with one of the poorest prognoses. Delta-24-RGD and ONC201 have shown promising efficacy as single agents for these tumors. However, the combination of both agents has not been evaluated. METHODS: The production of functional viruses was assessed by immunoblotting and replication assays. The antitumor effect was evaluated in a panel of human and murine pHGG and DMG cell lines. RNAseq, the seahorse stress test, mitochondrial DNA content, and γH2A.X immunofluorescence were used to perform mechanistic studies. Mouse models of both diseases were used to assess the efficacy of the combination in vivo. The tumor immune microenvironment was evaluated using flow cytometry, RNAseq and multiplexed immunofluorescence staining. RESULTS: The Delta-24-RGD/ONC201 combination did not affect the virus replication capability in human pHGG and DMG models in vitro. Cytotoxicity analysis showed that the combination treatment was either synergistic or additive. Mechanistically, the combination treatment increased nuclear DNA damage and maintained the metabolic perturbation and mitochondrial damage caused by each agent alone. Delta-24-RGD/ONC201 cotreatment extended the overall survival of mice implanted with human and murine pHGG and DMG cells, independent of H3 mutation status and location. Finally, combination treatment in murine DMG models revealed a reshaping of the tumor microenvironment to a proinflammatory phenotype. CONCLUSIONS: The Delta-24-RGD/ONC201 combination improved the efficacy compared to each agent alone in in vitro and in vivo models by potentiating nuclear DNA damage and in turn improving the antitumor (immune) response to each agent alone.

Intratumoral co-injection of NK cells and NKG2A-neutralizing monoclonal antibodies.

NK-cell reactivity against cancer is conceivably suppressed in the tumor microenvironment by the interaction of the inhibitory receptor NKG2A with the non-classical MHC-I molecules HLA-E in humans or Qa-1b in mice. We found that intratumoral delivery of NK cells attains significant therapeutic effects only if co-injected with anti-NKG2A and anti-Qa-1b blocking monoclonal antibodies against solid mouse tumor models. Such therapeutic activity was contingent on endogenous CD8 T cells and type-1 conventional dendritic cells (cDC1). Moreover, the anti-tumor effects were enhanced upon combination with systemic anti-PD-1 mAb treatment and achieved partial abscopal efficacy against distant non-injected tumors. In xenografted mice bearing HLA-E-expressing human cancer cells, intratumoral co-injection of activated allogeneic human NK cells and clinical-grade anti-NKG2A mAb (monalizumab) synergistically achieved therapeutic effects. In conclusion, these studies provide evidence for the clinical potential of intratumoral NK cell-based immunotherapies that exert their anti-tumor efficacy as a result of eliciting endogenous T-cell responses.

Intratumoral immunotherapy with mRNAs encoding chimeric protein constructs encompassing IL-12, CD137 agonists, and TGF-ß antagonists.

Intratumoral immunotherapy strategies for cancer based on interleukin-12 (IL-12)-encoding cDNA and mRNA are under clinical development in combination with anti-PD-(L)1 monoclonal antibodies. To make the most of these approaches, we have constructed chimeric mRNAs encoding single-chain IL-12 fused to single-chain fragment variable (scFv) antibodies that bind to transforming growth factor ß (TGF-ß) and CD137 (4-1BB). Several neutralizing TGF-ß agents and CD137 agonists are also undergoing early-phase clinical trials. To attain TGF-ß and CD137 binding by the constructions, we used bispecific tandem scFv antibodies (taFvs) derived from the specific 1D11 and 1D8 monoclonal antibodies (mAbs), respectively. Transfection of mRNAs encoding the chimeric constructs achieved functional expression of the proteins able to act on their targets. Upon mRNA intratumoral injections in the transplantable mouse cancer models CT26, MC38, and B16OVA, potent therapeutic effects were observed following repeated injections into the tumors. Efficacy was dependent on the number of CD8+ T cells able to recognize tumor antigens that infiltrated the malignant tissue. Although the abscopal effects on concomitant uninjected lesions were modest, such distant effects on untreated lesions were markedly increased when combined with systemic PD-1 blockade.

CD137 (4-1BB) requires physically associated cIAPs for signal transduction and antitumor effects.

CD137 (4-1BB) is a member of the TNFR family that mediates potent T cell costimulatory signals upon ligation by CD137L or agonist monoclonal antibodies (mAbs). CD137 agonists attain immunotherapeutic antitumor effects in cancer mouse models, and multiple agents of this kind are undergoing clinical trials. We show that cIAP1 and cIAP2 are physically associated with the CD137 signaling complex. Moreover, cIAPs are required for CD137 signaling toward the NF-κB and MAPK pathways and for costimulation of human and mouse T lymphocytes. Functional evidence was substantiated with SMAC mimetics that trigger cIAP degradation and by transfecting cIAP dominant-negative variants. Antitumor effects of agonist anti-CD137 mAbs are critically dependent on the integrity of cIAPs in cancer mouse models, and cIAPs are also required for signaling from CARs encompassing CD137's cytoplasmic tail.

Intratumoral neoadjuvant immunotherapy based on the BO-112 viral RNA mimetic.

BO-112 is a poly I:C-based viral mimetic that exerts anti-tumor efficacy when intratumorally delivered in mouse models. Intratumoral BO-112 synergizes in mice with systemic anti-PD-1 mAbs and this combination has attained efficacy in PD1-refractory melanoma patients. We sought to evaluate the anti-tumor efficacy of BO-112 pre-surgically applied in neoadjuvant settings to mouse models. We have observed that repeated intratumoral injections of BO-112 prior to surgical excision of the primary tumor significantly reduced tumor metastasis from orthotopically implanted 4T1-derived tumors and subcutaneous MC38-derived tumors in mice. Such effects were enhanced when combined with systemic anti-PD-1 mAb. The anti-tumor efficacy of this neoadjuvant immunotherapy approach depended on the presence of antigen-specific effector CD8 T cells and cDC1 antigen-presenting cells. Since BO-112 has been successful in phase-two clinical trials for metastatic melanoma, these results provide a strong rationale for translating this pre-surgical strategy into clinical settings, especially in combination with standard-of-care checkpoint inhibitors.

Killers on the loose: Immunotherapeutic strategies to improve NK cell-based therapy for cancer treatment.

Natural killer (NK) cells are innate lymphocytes that control tumor progression by not only directly killing cancer cells, but also by regulating other immune cells, helping to orchestrate a coordinated anti-tumor response. However, despite the tremendous potential that this cell type has, the clinical results obtained from diverse NK cell-based immunotherapeutic strategies have been, until recent years, rather modest. The intrinsic regulatory mechanisms that are involved in the control of their activation as well as the multiple mechanisms that tumor cells have developed to escape NK cell-mediated cytotoxicity likely account for the unsatisfactory clinical outcomes. The current approaches to improve long-term NK cell function are centered on modulating different molecules involved in both the activation and inhibition of NK cells, and the latest data seems to advocate for combining strategies that target multiple aspects of NK cell regulation. In this review, we summarize the different strategies (such as engineered NK cells, CAR-NK, NK cell immune engagers) that are currently being used to take advantage of this potent and complex immune cell.

Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies.

BACKGROUND: On the basis of efficacy in mouse tumor models, multiple CD137 (4-1BB) agonist agents are being preclinically and clinically developed. The costimulatory molecule CD137 is inducibly expressed as a transmembrane or as a soluble protein (sCD137). Moreover, the CD137 cytoplasmic signaling domain is a key part in approved chimeric antigen receptors (CARs). Reliable pharmacodynamic biomarkers for CD137 ligation and costimulation of T cells will facilitate clinical development of CD137 agonists in the clinic. METHODS: We used human and mouse CD8 T cells undergoing activation to measure CD137 transcription and protein expression levels determining both the membrane-bound and soluble forms. In tumor-bearing mice plasma sCD137 concentrations were monitored on treatment with agonist anti-CD137 monoclonal antibodies (mAbs). Human CD137 knock-in mice were treated with clinical-grade agonist anti-human CD137 mAb (Urelumab). Sequential plasma samples were collected from the first patients intratumorally treated with Urelumab in the INTRUST clinical trial. Anti-mesothelin CD137-encompassing CAR-transduced T cells were stimulated with mesothelin coated microbeads. sCD137 was measured by sandwich ELISA and Luminex. Flow cytometry was used to monitor CD137 surface expression. RESULTS: CD137 costimulation upregulates transcription and protein expression of CD137 itself including sCD137 in human and mouse CD8 T cells. Immunotherapy with anti-CD137 agonist mAb resulted in increased plasma sCD137 in mice bearing syngeneic tumors. sCD137 induction is also observed in human CD137 knock-in mice treated with Urelumab and in mice transiently humanized with T cells undergoing CD137 costimulation inside subcutaneously implanted Matrigel plugs. The CD137 signaling domain-containing CAR T cells readily released sCD137 and acquired CD137 surface expression on antigen recognition. Patients treated intratumorally with low dose Urelumab showed increased plasma concentrations of sCD137. CONCLUSION: sCD137 in plasma and CD137 surface expression can be used as quantitative parameters dynamically reflecting therapeutic costimulatory activity elicited by agonist CD137-targeted agents.

Three-dimensional colon cancer organoids model the response to CEA-CD3 T-cell engagers.

Rationale: The CEA-CD3 T cell bispecific antibody cibisatamab (CEA-TCB) is currently undergoing clinical trials. Here we study its performance against three-dimensional tumor organoids in cocultures with T cells as compared to a higher affinity CEACAM5-CD3 (CEACAM5-TCB) bispecific antibody using time-lapse confocal microscopy. Methods: Pre-labelled spheroids derived from colon cancer cell lines and primary organoids derived from four colorectal cancer surgical specimens, which expressed different graded levels of CEA, were exposed in cocultures to T lymphocytes. Cocultures were treated with CEA-CD3 T-cell engagers and were followed by live confocal microscopy. Caspase 3 activation detected in real-time was used as an indicator of tumor cell death. Co-cultures were also set up with autologous tumor-associated fibroblasts to test the co-stimulatory effect of a fibroblast activated protein (FAP)- targeted 4-1BBL bispecific antibody fusion protein currently undergoing clinical trials. Results: Tumor-cell killing of 3D colon carcinoma cultures was dependent on the levels of surface CEA expression, in such a way that the lower affinity agent (CEA-TCB) did not mediate killing by human preactivated T cells below a certain CEA expression threshold, while the high affinity construct (CEACAM5-TCB) remained active on the low CEA expressing organoids. Modelling heterogeneity in the levels of CEA expression by coculturing CEA high and low organoids showed measurable but weak bystander killing. Cocultures of tumor organoids, autologous fibroblasts and T cells allowed to observe a costimulatory effect of anti-FAP-4-1BBL both to release IFNγ and to attain more efficacious tumor cell killing. Conclusion: Three-dimensional tumor cocultures with T cells using live confocal microscopy provide suitable models to test the requirements for colon-cancer redirected killing as elicited by CEA-targeted T-cell engagers undergoing clinical trials and treatment allow combinations to be tested in a relevant preclinical system.

A Therapeutically Actionable Protumoral Axis of Cytokines Involving IL-8, TNFα, and IL-1ß.

Interleukin-8 (CXCL8) produced in the tumor microenvironment correlates with poor response to checkpoint inhibitors and is known to chemoattract and activate immunosuppressive myeloid leukocytes. In human cancer, IL8 mRNA levels correlate with IL1B and TNF transcripts. Both cytokines induced IL-8 functional expression from a broad variety of human cancer cell lines, primary colon carcinoma organoids, and fresh human tumor explants. Although IL8 is absent from the mouse genome, a similar murine axis in which TNFα and IL-1ß upregulate CXCL1 and CXCL2 in tumor cells was revealed. Furthermore, intratumoral injection of TNFα and IL-1ß induced IL-8 release from human malignant cells xenografted in immunodeficient mice. In all these cases, the clinically used TNFα blockers infliximab and etanercept or the IL-1ß inhibitor anakinra was able to interfere with this pathogenic cytokine loop. Finally, in paired plasma samples of patients with cancer undergoing TNFα blockade with infliximab in a clinical trial, reductions of circulating IL-8 were substantiated. SIGNIFICANCE: IL-8 attracts immunosuppressive protumor myeloid cells to the tumor microenvironment, and IL-8 levels correlate with poor response to checkpoint inhibitors. TNFα and IL-1ß are identified as major inducers of IL-8 expression on malignant cells across cancer types and models in a manner that is druggable with clinically available neutralizing agents. This article is highlighted in the In This Issue feature, p. 2007.

IL8, Neutrophils, and NETs in a Collusion against Cancer Immunity and Immunotherapy.

One of the most important mechanisms by which cancer fosters its own development is the generation of an immune microenvironment that inhibits or impairs antitumor immune responses. A cancer permissive immune microenvironment is present in a large proportion of the patients with cancer who do not respond to immunotherapy approaches intended to trigger preexisting antitumor immune responses, for instance, immune checkpoint blockade. High circulating levels of IL8 in patients with cancer quite accurately predict those who will not benefit from checkpoint-based immunotherapy. IL8 has been reported to favor cancer progression and metastases via different mechanisms, including proangiogenesis and the maintenance of cancer stem cells, but its ability to attract and functionally modulate neutrophils and macrophages is arguably one of the most important factors. IL8 does not only recruit neutrophils to tumor lesions, but also triggers the extrusion of neutrophil extracellular traps (NET). The relevance and mechanisms underlying the contribution of both neutrophils and NETs to cancer development and progression are starting to be uncovered and include both direct effects on cancer cells and changes in the tumor microenvironment, such as facilitating metastasis, awakening micrometastases from dormancy, and facilitating escape from cytotoxic immune cells. Blockade of IL8 or its receptors (CXCR1 and CXCR2) is being pursued in drug development, and clinical trials alone or in combination with anti-PD-L1 checkpoint inhibitors are already ongoing.

CD137 Costimulation Counteracts TGFß Inhibition of NK-cell Antitumor Function.

Enhancing natural killer (NK) cell-based cancer immunotherapy by overcoming immunosuppression is an area of intensive research. Here, we have demonstrated that the anti-CD137 agonist urelumab can overcome TGFß-mediated inhibition of human NK-cell proliferation and antitumor function. Transcriptomic, immunophenotypic, and functional analyses showed that CD137 costimulation modified the transcriptional program induced by TGFß on human NK cells by rescuing their proliferation in response to IL2, preserving their expression of activating receptors (NKG2D) and effector molecules (granzyme B, IFNγ) while allowing the acquisition of tumor-homing/retention features (CXCR3, CD103). Activated NK cells cultured in the presence of TGFß1 and CD137 agonist recovered CCL5 and IFNγ secretion and showed enhanced direct and antibody-dependent cytotoxicity upon restimulation with cancer cells. Trastuzumab treatment of fresh breast carcinoma-derived multicellular cultures induced CD137 expression on tumor-infiltrating CD16+ NK cells, enabling the action of urelumab, which fostered tumor-infiltrating NK cells and recapitulated the enhancement of CCL5 and IFNγ production. Bioinformatic analysis pointed to IFNG as the driver of the association between NK cells and clinical response to trastuzumab in patients with HER2-positive primary breast cancer, highlighting the translational relevance of the CD137 costimulatory axis for enhancing IFNγ production. Our data reveals CD137 as a targetable checkpoint for overturning TGFß constraints on NK-cell antitumor responses.

An Fc-free EGFR-specific 4-1BB-agonistic Trimerbody Displays Broad Antitumor Activity in Humanized Murine Cancer Models without Toxicity.

PURPOSE: The induction of 4-1BB signaling by agonistic antibodies can drive the activation and proliferation of effector T cells and thereby enhance a T-cell-mediated antitumor response. Systemic administration of anti-4-1BB-agonistic IgGs, although effective preclinically, has not advanced in clinical development due to their severe hepatotoxicity. EXPERIMENTAL DESIGN: Here, we generated a humanized EGFR-specific 4-1BB-agonistic trimerbody, which replaces the IgG Fc region with a human collagen homotrimerization domain. It was characterized by structural analysis and in vitro functional studies. We also assessed pharmacokinetics, antitumor efficacy, and toxicity in vivo. RESULTS: In the presence of a T-cell receptor signal, the trimerbody provided potent T-cell costimulation that was strictly dependent on 4-1BB hyperclustering at the point of contact with a tumor antigen-displaying cell surface. It exhibits significant antitumor activity in vivo, without hepatotoxicity, in a wide range of human tumors including colorectal and breast cancer cell-derived xenografts, and non-small cell lung cancer patient-derived xenografts associated with increased tumor-infiltrating CD8+ T cells. The combination of the trimerbody with a PD-L1 blocker led to increased IFNγ secretion in vitro and resulted in tumor regression in humanized mice bearing aggressive triple-negative breast cancer. CONCLUSIONS: These results demonstrate the nontoxic broad antitumor activity of humanized Fc-free tumor-specific 4-1BB-agonistic trimerbodies and their synergy with checkpoint blockers, which may provide a way to elicit responses in most patients with cancer while avoiding Fc-mediated adverse reactions.

In vivo depletion of DC impairs the anti-tumor effect of agonistic anti-CD137 mAb.

Anti-CD137 mAb are capable of inducing tumor rejection in several syngeneic murine tumor models and are undergoing clinical trials for cancer. The anti-tumor effect involves co-stimulation of tumor-specific CD8(+) T cells. Whether antigen cross-presenting DC are required for the efficacy of anti-CD137 mAb treatment has never been examined. Here we show that the administration of anti-CD137 mAb eradicates EG7-OVA tumors by a strictly CD8beta(+) T-cell-dependent mechanism that correlates with increased CTL activity. Ex vivo analyses to determine the identity of the draining lymph node cell type responsible for tumor antigen cross-presentation revealed that CD11c(+) cells, most likely DC, are the main players in this tumor model. A minute number of tumor cells, revealed by the presence of OVA cDNA, reach tumor-draining lymph nodes. Direct antigen presentation by tumor cells themselves also participates in anti-OVA CTL induction. Using CD11c diphtheria toxin receptor-green fluorescent protein-->C57BL/6 BM chimeric mice, which allow for sustained ablation of DC with diphtheria toxin, we confirmed the involvement of DC in tumor antigen cross-presentation in CTL induction against OVA(257-264) epitope and in the antitumor efficacy induced by anti-CD137 mAb.

Treatment with anti-CD137 mAbs causes intense accumulations of liver T cells without selective antitumor immunotherapeutic effects in this organ.

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.

Dietary total antioxidant capacity and obesity in children and adolescents.

BACKGROUND: Dietary antioxidant intake has been suggested to protect against oxidative damage and related clinical complications. The aim of the present study was to assess the potential relationships between the dietary total antioxidant capacity (TAC) and obesity-related features in children and adolescents. MATERIALS AND METHODS: Anthropometric variables from 369 children and adolescents were measured (184 obese and 185 control). A validated food-frequency questionnaire was used to calculate the TAC and the daily nutrient and energy intake. RESULTS: Dietary TAC showed positive associations with fiber, folic acid, magnesium, and vitamins A, C and E. The body mass index, standard deviation score of body mass index and total body fat were inversely associated with dietary TAC only in obese subjects. CONCLUSION: These data suggest that dietary TAC may be a potential indicator of the risk to develop obesity-related features and could be considered a useful method in assessing antioxidant intake.

Rapid isolation and enrichment of mouse NK cells for experimental purposes.

Natural killer (NK) cells have shown to play a critical, but as yet poorly defined, role in the process by which the immune system controls tumor progression. Indeed, NK cell-based immunotherapy, particularly NK cell adoptive transfer therapy, has become a very attractive cancer weapon against multiple types of cancers such as metastatic and hematological cancers. Unfortunately, the implementation of these therapies has been challenged by the existence of immunosuppression mechanisms that have prevented NK cell functionality. Additionally, the development of protocols to obtain purified and functional NK cells has faced some difficulties due to the limitations in the numbers of cells that can be obtained and the development of an exhaustion phenotype with impaired proliferative and functional capabilities during lengthy ex vivo NK cell expansion protocols. Thus, the development of new strategies to obtain a rapid expansion of highly functional NK cells without the appearance of exhaustion is still much needed. This is particularly true in the case of mouse NK cells, a surrogate commonly used to evaluate NK cell biology and human NK cell-based immunotherapeutic alternatives. Here, we describe a feasible and rapid protocol to produce strongly activated mouse NK cells in vivo taking advantage of the hydrodynamic delivery of a plasmid that contains interleukin-15, a cytokine known to cause NK cell expansion and activation, fused with the binding domain of the IL-15Rα ("sushi" domain) and apolipoprotein A-I.