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-?B 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.

Immune checkpoint-inhibitor combinations are the best therapeutic option for advanced hepatocellular car-cinoma (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 addi-tion 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 reg-imens 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.

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 TNF alpha and IL-1 beta upregulate CXCL1 and CXCL2 in tumor cells was revealed. Furthermore, intratumoral injection of TNF alpha and IL-1 beta induced IL-8 release from human malignant cells xenografted in immunodeficient mice. In all these cases, the clinically used TNF alpha blockers infliximab and etanercept or the IL-1 beta inhibitor anakinra was able to interfere with this pathogenic cytokine loop. Finally, in paired plasma samples of patients with cancer undergoing TNF alpha 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. TNF alpha and IL-1 beta 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.

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.

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.

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-kappa B 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. Costimulation has been shown to be required for optimal activation of T cells and it could be delivered either in trans with respect to the source of CD3-TCR ligation or in cis on the same cell. Here the authors show that CD137 costimulation is more effective when delivered in cis to enhance T cell proliferation and activation.

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.

Background The immune response to cancer is often conceptualized with the cancer immunity cycle. An essential step in this interpretation is that antigens released by dying tumors are presented by dendritic cells to naive or memory T cells in the tumor-draining lymph nodes. Whether tumor cell death resulting from cytotoxicity, as mediated by T cells or natural killer (NK) lymphocytes, is actually immunogenic currently remains unknown. Methods In this study, tumor cells were killed by antigen-specific T-cell receptor (TCR) transgenic CD8 T cells or activated NK cells. Immunogenic cell death was studied analyzing the membrane exposure of calreticulin and the release of high mobility group box 1 (HMGB1) by the dying tumor cells. Furthermore, the potential immunogenicity of the tumor cell debris was evaluated in immunocompetent mice challenged with an unrelated tumor sharing only one tumor-associated antigen and by class I major histocompatibility complex (MHC)-multimer stainings. Mice deficient in Batf3, Ifnar1 and Sting1 were used to study mechanistic requirements. Results We observe in cocultures of tumor cells and effector cytotoxic cells, the presence of markers of immunogenic cell death such as calreticulin exposure and soluble HMGB1 protein. Ovalbumin (OVA)-transfected MC38 colon cancer cells, exogenously pulsed to present the gp100 epitope are killed in culture by mouse gp100-specific TCR transgenic CD8 T cells. Immunization of mice with the resulting destroyed cells induces epitope spreading as observed by detection of OVA-specific T cells by MHC multimer staining and rejection of OVA(+) EG7 lymphoma cells. Similar results were observed in mice immunized with cell debris generated by NK-cell mediated cytotoxicity. Mice deficient in Batf3-dependent dendritic cells (conventional dendritic cells type 1, cDC1) fail to develop an anti-OVA response when immunized with tumor cells killed by cytotoxic lymphocytes. In line with this, cultured cDC1 dendritic cells uptake and can readily cross-present antigen from cytotoxicity-killed tumor cells to cognate CD8(+) T lymphocytes. Conclusion These results support that an ongoing cytotoxic antitumor immune response can lead to immunogenic tumor 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.

Radiotherapy can be synergistically combined with immunotherapy in mouse models, extending its efficacious effects outside of the irradiated field (abscopal effects). We previously reported that a regimen encompassing local radiotherapy in combination with anti-CD137 plus anti-PD-1 mAbs achieves potent abscopal effects against syngeneic transplanted murine tumors up to a certain tumor size. Knowing that TGF beta expression or activation increases in irradiated tissues, we tested whether TGF beta blockade may further enhance abscopal effects in conjunction with the anti-PD-1 plus anti-CD137 mAb combination. Indeed, TGF beta blockade with 1D11, a TGF beta-neutralizing mAb, markedly enhanced abscopal effects and overall treatment efficacy against subcutaneous tumors of either 4T1 breast cancer cells or large MC38 colorectal tumors. Increases in CD8 T cells infiltrating the nonirradiated lesion were documented upon combined treatment, which intensely expressed Granzyme-B as an indicator of cytotoxic effector capability. Interestingly, tumor tissue but not healthy tissue irradiation results in the presence of higher concentrations of TGF beta in the nonirradiated contralateral tumor that showed smad2/3 phosphorylation increases in infiltrating CD8 T cells. In conclusion, radiotherapy-induced TGF beta hampers abscopal efficacy even upon combination with a potent immunotherapy regimen. Therefore, TGF beta blockade in combination with radioimmunotherapy results in greater efficacy.