CXCR1 and CXCR2 Chemokine Receptor Agonists Produced by Tumors Induce Neutrophil Extracellular Traps that Interfere with Immune Cytotoxicity.

Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+ T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.

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.

Focusing and sustaining the antitumor CTL effector killer response by agonist anti-CD137 mAb.

Cancer immunotherapy is undergoing significant progress due to recent clinical successes by refined adoptive T-cell transfer and immunostimulatory monoclonal Ab (mAbs). B16F10-derived OVA-expressing mouse melanomas resist curative immunotherapy with either adoptive transfer of activated anti-OVA OT1 CTLs or agonist anti-CD137 (4-1BB) mAb. However, when acting in synergistic combination, these treatments consistently achieve tumor eradication. Tumor-infiltrating lymphocytes that accomplish tumor rejection exhibit enhanced effector functions in both transferred OT-1 and endogenous cytotoxic T lymphocytes (CTLs). This is consistent with higher levels of expression of eomesodermin in transferred and endogenous CTLs and with intravital live-cell two-photon microscopy evidence for more efficacious CTL-mediated tumor cell killing. Anti-CD137 mAb treatment resulted in prolonged intratumor persistence of the OT1 CTL-effector cells and improved function with focused and confined interaction kinetics of OT-1 CTL with target cells and increased apoptosis induction lasting up to six days postadoptive transfer. The synergy of adoptive T-cell therapy and agonist anti-CD137 mAb thus results from in vivo enhancement and sustainment of effector functions.

Deciphering CD137 (4-1BB) signaling in T-cell costimulation for translation into successful cancer immunotherapy.

CD137 (4-1BB, TNF-receptor superfamily 9) is a surface glycoprotein of the TNFR family which can be induced on a variety of leukocyte subsets. On T and NK cells, CD137 is expressed following activation and, if ligated by its natural ligand (CD137L), conveys polyubiquitination-mediated signals via TNF receptor associated factor 2 that inhibit apoptosis, while enhancing proliferation and effector functions. CD137 thus behaves as a bona fide inducible costimulatory molecule. These functional properties of CD137 can be exploited in cancer immunotherapy by systemic administration of agonist monoclonal antibodies, which increase anticancer CTLs and enhance NK-cell-mediated antibody-dependent cell-mediated cytotoxicity. Reportedly, anti-CD137 mAb and adoptive T-cell therapy strongly synergize, since (i) CD137 expression can be used to select the T cells endowed with the best activities against the tumor, (ii) costimulation of the lymphocyte cultures to be used in adoptive T-cell therapy can be done with CD137 agonist antibodies or CD137L, and (iii) synergistic effects upon coadministration of T cells and antibodies are readily observed in mouse models. Furthermore, the signaling cytoplasmic tail of CD137 is a key component of anti-CD19 chimeric antigen receptors that are used to redirect T cells against leukemia and lymphoma in the clinic. Ongoing phase II clinical trials with agonist antibodies and the presence of CD137 sequence in these successful chimeric antigen receptors highlight the importance of CD137 in oncoimmunology.

T cell costimulation with anti-CD137 monoclonal antibodies is mediated by K63-polyubiquitin-dependent signals from endosomes.

Agonist anti-CD137 (4-1BB) mAbs enhance CD8-mediated antitumor immunity. Agonist anti-human CD137 mAbs binding to four distinct epitopes on the CD137 glycoprotein costimulated T cell activation irrespective of the engaged epitope or its interference with CD137L binding. CD137 perturbation with all these agonist mAbs resulted in Ag and Ab internalization toward an endosomal vesicular compartment. Internalization was observed in activated T lymphocytes from humans and mice, not only in culture but also in Ab-injected living animals. These in vivo experiments were carried out upon systemic i.v. injections with anti-CD137 mAbs and showed CD137 internalization in tumor-infiltrating lymphocytes and in activated human T cells transferred to immunodeficient mice. Efficient CD137 internalization required K63 polyubiquitination and endocytosed CD137-containing vesicles recruited TNFR-associated factor (TRAF) 2 and were decorated with K63 polyubiquitins. CD137 stimulation activates NF-κB through a K63-linked polyubiquitination-dependent route, and CD137-associated TRAF2 becomes K63 polyubiquitinated. Consistent with a role for TRAF2 in CD137 signaling, transgenic mice functionally deficient in TRAF2 showed delayed immunotherapeutic activity of anti-CD137 mAbs. As a whole, these findings advance our knowledge of the mechanisms of action of anti-CD137 immunostimulatory mAbs such as those currently undergoing clinical trials in cancer patients.

Short-term cultured tumor fragments to study immunotherapy combinations based on CD137 (4-1BB) agonism.

Biomarkers for cancer immunotherapy are an unmet medical need. The group of Daniela Thommen at the NKI recently reported on novel methodologies based on short-term cultures of patient-derived tumor fragments whose cytokine concentrations in the supernatants and activation markers on infiltrating T cells were associated with clinical response to PD-1 blockade. We set up a similar culture technology with tumor-derived fragments using mouse tumors transplanted into syngeneic immunocompetent mice to test an agonist anti-CD137 mAb and its combinations with anti-PD-1 and/or anti-TGF-ß. Increases in IFNγ concentrations in the tissue culture supernatants were detected upon in-culture activation with the anti-CD137 and anti-PD-1 mAb combinations or concanavalin A as a positive control. No other cytokine from a wide array was informative of stimulation with these mAbs. Interestingly, increases in Ki67 and other activation markers were substantiated in lymphocytes from cell suspensions gathered at the end of 72 h cultures. In mice bearing bilateral tumors in which one was excised prior to in vivo anti-CD137 + anti-PD-1 treatment to perform the fragment culture evaluation, no association was found between IFNγ production from the fragments and the in vivo therapeutic outcome in the non-resected contralateral tumors. The experimental system permitted freezing and thawing of the fragments with similar functional outcomes. Using a series of patient-derived tumor fragments from excised solid malignancies, we showed IFNγ production in a fraction of the studied cases, that was conserved in frozen/thawed fragments. The small tumor fragment culture technique seems suitable to preclinically explore immunotherapy combinations.

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.

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.

Synergistic effects of combined immunotherapy strategies in a model of multifocal hepatocellular carcinoma.

Immune checkpoint-inhibitor combinations are the best therapeutic option for advanced hepatocellular carcinoma (HCC) patients, but improvements in efficacy are needed to improve response rates. We develop a multifocal HCC model to test immunotherapies by introducing c-myc using hydrodynamic gene transfer along with CRISPR-Cas9-mediated disruption of p53 in mouse hepatocytes. Additionally, induced co-expression of luciferase, EGFP, and the melanosomal antigen gp100 facilitates studies on the underlying immunological mechanisms. We show that treatment of the mice with a combination of anti-CTLA-4 + anti-PD1 mAbs results in partial clearance of the tumor with an improvement in survival. However, the addition of either recombinant IL-2 or an anti-CD137 mAb markedly improves both outcomes in these mice. Combining tumor-specific adoptive T cell therapy to the aCTLA-4/aPD1/rIL2 or aCTLA-4/aPD1/aCD137 regimens enhances efficacy in a synergistic manner. As shown by multiplex tissue immunofluorescence and intravital microscopy, combined immunotherapy treatments enhance T cell infiltration and the intratumoral performance of T lymphocytes.

Intratumoral Gene Transfer of mRNAs Encoding IL12 in Combination with Decoy-Resistant IL18 Improves Local and Systemic Antitumor Immunity.

IL12-based local gene therapy of cancer constitutes an active area of clinical research using plasmids, mRNAs, and viral vectors. To improve antitumor effects, we have experimentally tested the combination of mRNA constructs encoding IL12 and IL18. Moreover, we have used a form of IL18 [decoy-resistant IL18 (DR-18)] which has preserved bioactivity but does not bind to the IL18 binding protein decoy receptor. Both cytokines dramatically synergize to induce IFNγ release from mouse splenocytes, and, if systemically cotransferred to the liver, they mediate lethal toxicity. However, if given intratumorally to B16OVA tumor-bearing mice, the combination attains efficacy against the directly treated tumor and moderate tumor-delaying activity on distant noninjected lesions. Cotreatment was conducive to the presence of more activated CD8+ T cells in the treated and noninjected tumors. In keeping with these findings, the efficacy of treatment was contingent on the integrity of CD8+ T cells and cDC1 dendritic cells in the treated mice. Furthermore, efficacy of IL12 plus DR-18 local mRNA coinjection against distant concomitant tumors could be enhanced upon combination with anti-PD-1 mAb systemic treatment, thus defining a feasible synergistic immunotherapy strategy.

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.

Two cell line models to study multiorganic metastasis and immunotherapy in lung squamous cell carcinoma.

There is a paucity of adequate mouse models and cell lines available to study lung squamous cell carcinoma (LUSC). We have generated and characterized two models of phenotypically different transplantable LUSC cell lines, i.e. UN-SCC679 and UN-SCC680, derived from A/J mice that had been chemically induced with N-nitroso-tris-chloroethylurea (NTCU). Furthermore, we genetically characterized and compared both LUSC cell lines by performing whole-exome and RNA sequencing. These experiments revealed similar genetic and transcriptomic patterns that may correspond to the classic LUSC human subtype. In addition, we compared the immune landscape generated by both tumor cells lines in vivo and assessed their response to immune checkpoint inhibition. The differences between the two cell lines are a good model for the remarkable heterogeneity of human squamous cell carcinoma. Study of the metastatic potential of these models revealed that both cell lines represent the organotropism of LUSC in humans, i.e. affinity to the brain, bones, liver and adrenal glands. In summary, we have generated valuable cell line tools for LUSC research, which recapitulates the complexity of the human disease.

Depletion of Conventional Type-1 Dendritic Cells in Established Tumors Suppresses Immunotherapy Efficacy.

The ability of conventional type-1 dendritic cells (cDC1) to cross-present tumor antigens to CD8+ T cells is critical for the induction of antitumor CTLs. Mice that are constitutively deficient in cDC1 cells have been reported to fail to respond to immunotherapy strategies based on checkpoint inhibitors. However, further work is needed to clarify the precise time during immunotherapy treatment that cDC1 cells are required for the beneficial effect of treatment. Here, we used a refined XCR1-DTR-Venus transgenic mouse model to acutely deplete cDC1 cells and trace their behavior using intravital microscopy. Diphtheria toxin-mediated cDC1 depletion prior to immunotherapy treatment with anti-PD-1 and/or anti-CD137 immunostimulatory mAbs completely ablated antitumor efficacy. The efficacy of adoptive T-cell therapy was also hampered by prior cDC1 depletion. After the onset of immunotherapy treatment, depletion of cDC1s only moderately reduced the therapeutic efficacy of anti-PD-1 and anti-CD137 mAbs. Intravital microscopy of liver-engrafted tumors revealed changes in the intratumoral behavior of cDC1 cells in mice receiving immunotherapy, and treatment with diphtheria toxin to deplete cDC1s impaired tumor T-cell infiltration and function. These results reveal that the functional integrity of the cDC1 compartment is required at the onset of various immunotherapies to successfully treat established tumors. SIGNIFICANCE: These findings reveal the intratumoral behavior of cDC1 dendritic cells in transgenic mouse models and demonstrate that the efficacy of immunotherapy regimens is precluded by elimination of these cells.

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.

Intratumoral injection of interferon-α and systemic delivery of agonist anti-CD137 monoclonal antibodies synergize for immunotherapy.

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.

CD137 (4-1BB) costimulation of CD8+ T cells is more potent when provided in cis than in trans with respect to CD3-TCR stimulation.

CD137 (4-1BB; TNFSR9) is an activation-induced surface receptor that through costimulation effects provide antigen-primed T cells with augmented survival, proliferation and effector functions as well as metabolic advantages. These immunobiological mechanisms are being utilised for cancer immunotherapy with agonist CD137-binding and crosslinking-inducing agents that elicit CD137 intracellular signaling. In this study, side-by-side comparisons show that provision of CD137 costimulation in-cis with regard to the TCR-CD3-ligating cell is superior to that provided in-trans in terms of T cell activation, proliferation, survival, cytokine secretion and mitochondrial fitness in mouse and human. Cis ligation of CD137 relative to the TCR-CD3 complex results in more intense canonical and non-canonical NF-κB signaling and provides a more robust induction of cell cycle and DNA damage repair gene expression programs. Here we report that the superiority of cis versus trans CD137-costimulation is readily observed in vivo and is relevant for understanding the immunotherapeutic effects of CAR T cells and CD137 agonistic therapies currently undergoing clinical trials, which may provide costimulation either in cis or in trans.

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.

Human CD8 T cells are susceptible to TNF-mediated activation-induced cell death.

Activation-induced cell death (AICD) is a complex immunoregulatory mechanism that causes the demise of a fraction of T-lymphocytes upon antigen-driven activation. In the present study we investigated the direct role of TNF in AICD of CD8 T lymphocytes. Methods: Human peripheral mononuclear cells were isolated from healthy donors and fresh tumor-infiltrating lymphocytes were obtained from cancer patients undergoing surgery. T cells were activated with anti-CD3/CD28 mAbs or with a pool of virus peptides, in combination with clinical-grade TNF blocking agents. Results: A portion of CD8 T cells undergoes apoptosis upon CD3/CD28 activation in a manner that is partially prevented by the clinically used anti-TNF agents infliximab and etanercept. TNF-mediated AICD was also observed upon activation of virus-specific CD8 T cells and tumor-infiltrating CD8 T lymphocytes. The mechanism of TNF-driven T cell death involves TNFR2 and production of mitochondrial oxygen free radicals which damage DNA. Conclusion: The use of TNF blocking agents reduces oxidative stress, hyperpolarization of mitochondria, and the generation of DNA damage in CD8 T celss undergoing activation. The fact that TNF mediates AICD in human tumor-reactive CD8 T cells suggests that the use of TNF-blocking agents can be exploited in immunotherapy strategies.